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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. Targeting SR-BI for Cancer Diagnostics, Imaging and Therapy

    PubMed Central

    Rajora, Maneesha A.; Zheng, Gang

    2016-01-01

    Scavenger receptor class B type I (SR-BI) plays an important role in trafficking cholesteryl esters between the core of high density lipoprotein and the liver. Interestingly, this integral membrane protein receptor is also implicated in the metabolism of cholesterol by cancer cells, whereby overexpression of SR-BI has been observed in a number of tumors and cancer cell lines, including breast and prostate cancers. Consequently, SR-BI has recently gained attention as a cancer biomarker and exciting target for the direct cytosolic delivery of therapeutic agents. This brief review highlights these key developments in SR-BI-targeted cancer therapies and imaging probes. Special attention is given to the exploration of high density lipoprotein nanomimetic platforms that take advantage of upregulated SR-BI expression to facilitate targeted drug-delivery and cancer diagnostics, and promising future directions in the development of these agents. PMID:27729859

  7. Intestinal SR-BI does not impact cholesterol absorption or transintestinal cholesterol efflux in mice.

    PubMed

    Bura, Kanwardeep S; Lord, Caleb; Marshall, Stephanie; McDaniel, Allison; Thomas, Gwyn; Warrier, Manya; Zhang, Jun; Davis, Matthew A; Sawyer, Janet K; Shah, Ramesh; Wilson, Martha D; Dikkers, Arne; Tietge, Uwe J F; Collet, Xavier; Rudel, Lawrence L; Temel, Ryan E; Brown, J Mark

    2013-06-01

    Reverse cholesterol transport (RCT) can proceed through the classic hepatobiliary route or through the nonbiliary transintestinal cholesterol efflux (TICE) pathway. Scavenger receptor class B type I (SR-BI) plays a critical role in the classic hepatobiliary route of RCT. However, the role of SR-BI in TICE has not been studied. To examine the role of intestinal SR-BI in TICE, sterol balance was measured in control mice and mice transgenically overexpressing SR-BI in the proximal small intestine (SR-BI(hApoCIII-ApoAIV-Tg)). SR-BI(hApoCIII-ApoAIV-Tg) mice had significantly lower plasma cholesterol levels compared with wild-type controls, yet SR-BI(hApoCIII-ApoAIV-Tg) mice had normal fractional cholesterol absorption and fecal neutral sterol excretion. Both in the absence or presence of ezetimibe, intestinal SR-BI overexpression had no impact on the amount of cholesterol excreted in the feces. To specifically study effects of intestinal SR-BI on TICE we crossed SR-BI(hApoCIII-ApoAIV-Tg) mice into a mouse model that preferentially utilized the TICE pathway for RCT (Niemann-Pick C1-like 1 liver transgenic), and likewise found no alterations in cholesterol absorption or fecal sterol excretion. Finally, mice lacking SR-BI in all tissues also exhibited normal cholesterol absorption and fecal cholesterol disposal. Collectively, these results suggest that SR-BI is not rate limiting for intestinal cholesterol absorption or for fecal neutral sterol loss through the TICE pathway.

  8. SR-BI: Linking Cholesterol and Lipoprotein Metabolism with Breast and Prostate Cancer

    PubMed Central

    Gutierrez-Pajares, Jorge L.; Ben Hassen, Céline; Chevalier, Stéphan; Frank, Philippe G.

    2016-01-01

    Studies have demonstrated the significant role of cholesterol and lipoprotein metabolism in the progression of cancer. The SCARB1 gene encodes the scavenger receptor class B type I (SR-BI), which is an 82-kDa glycoprotein with two transmembrane domains separated by a large extracellular loop. SR-BI plays an important role in the regulation of cholesterol exchange between cells and high-density lipoproteins. Accordingly, hepatic SR-BI has been shown to play an essential role in the regulation of the reverse cholesterol transport pathway, which promotes the removal and excretion of excess body cholesterol. In the context of atherosclerosis, SR-BI has been implicated in the regulation of intracellular signaling, lipid accumulation, foam cell formation, and cellular apoptosis. Furthermore, since lipid metabolism is a relevant target for cancer treatment, recent studies have focused on examining the role of SR-BI in this pathology. While signaling pathways have initially been explored in non-tumoral cells, studies with cancer cells have now demonstrated SR-BI's function in tumor progression. In this review, we will discuss the role of SR-BI during tumor development and malignant progression. In addition, we will provide insights into the transcriptional and post-transcriptional regulation of the SCARB1 gene. Overall, studying the role of SR-BI in tumor development and progression should allow us to gain useful information for the development of new therapeutic strategies. PMID:27774064

  9. 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

  10. Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling and reduces atherosclerotic lesion necrosis[S

    PubMed Central

    Tao, Huan; Yancey, Patricia G.; Babaev, Vladimir R.; Blakemore, John L.; Zhang, Youmin; Ding, Lei; Fazio, Sergio; Linton, MacRae F.

    2015-01-01

    Macrophage apoptosis and efferocytosis are key determinants of atherosclerotic plaque inflammation and necrosis. Bone marrow transplantation studies in ApoE- and LDLR-deficient mice revealed that hematopoietic scavenger receptor class B type I (SR-BI) deficiency results in severely defective efferocytosis in mouse atherosclerotic lesions, resulting in a 17-fold higher ratio of free to macrophage-associated dead cells in lesions containing SR-BI−/− cells, 5-fold more necrosis, 65.2% less lesional collagen content, nearly 7-fold higher dead cell accumulation, and 2-fold larger lesion area. Hematopoietic SR-BI deletion elicited a maladaptive inflammatory response [higher interleukin (IL)-1β, IL-6, and TNF-α lower IL-10 and transforming growth factor β]. Efferocytosis of apoptotic thymocytes was reduced by 64% in SR-BI−/− versus WT macrophages, both in vitro and in vivo. In response to apoptotic cells, macrophage SR-BI bound with phosphatidylserine and induced Src phosphorylation and cell membrane recruitment, which led to downstream activation of phosphoinositide 3-kinase (PI3K) and Ras-related C3 botulinum toxin substrate 1 (Rac1) for engulfment and clearance of apoptotic cells, as inhibition of Src decreased PI3K, Rac1-GTP, and efferocytosis in WT cells. Pharmacological inhibition of Rac1 reduced macrophage efferocytosis in a SR-BI-dependent fashion, and activation of Rac1 corrected the defective efferocytosis in SR-BI−/− macrophages. Thus, deficiency of macrophage SR-BI promotes defective efferocytosis signaling via the Src/PI3K/Rac1 pathway, resulting in increased plaque size, necrosis, and inflammation. PMID:26059978

  11. SR-BI in Bone Marrow Derived Cells Protects Mice from Diet Induced Coronary Artery Atherosclerosis and Myocardial Infarction

    PubMed Central

    Pei, Ying; Chen, Xing; Aboutouk, Dina; Fuller, Mark T.; Dadoo, Omid; Yu, Pei; White, Elizabeth J.; Igdoura, Suleiman A.; Trigatti, Bernardo L.

    2013-01-01

    SR-BI deficient mice that are also hypomorphic for apolipoprotein E expression develop diet induced occlusive coronary artery atherosclerosis, myocardial infarction and early death. To test the role of SR-BI in bone marrow derived cells, we used bone marrow transplantation to generate SR-BI-null; apoE-hypomorphic mice in which SR-BI expression was restored solely in bone marrow derived cells. SR-BI-null; apoE-hypomorphic mice were transplanted with SR-BI+/+apoE-hypomorphic, or control, autologous SR-BI-null; apoE-hypomorphic bone marrow. Four weeks later, mice were fed a high-fat, high-cholesterol, cholate-containing diet to induce coronary artery atherosclerosis. Mice transplanted with autologous bone marrow developed extensive aortic atherosclerosis and severe occlusive coronary artery atherosclerosis after 4 weeks of feeding. This was accompanied by myocardial fibrosis and increased heart weights. In contrast, restoration of SR-BI expression in bone marrow derived-cells reduced diet induced aortic and coronary artery atherosclerosis, myocardial fibrosis and the increase in heart weights in SR-BI-null; apoE-hypomorphic mice. Restoration of SR-BI in bone marrow derived cells did not, however, affect steady state lipoprotein cholesterol levels, but did reduce plasma levels of IL-6. Monocytes from SR-BI-null mice exhibited a greater capacity to bind to VCAM-1 and ICAM-1 than those from SR-BI+/+ mice. Furthermore, restoration of SR-BI expression in bone marrow derived cells attenuated monocyte recruitment into atherosclerotic plaques in mice fed high fat, high cholesterol cholate containing diet. These data demonstrate directly that SR-BI in bone marrow-derived cells protects against both aortic and CA atherosclerosis. PMID:23967310

  12. 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.

  13. Distinct surveillance pathway for immunopathology during acute infection via autophagy and SR-BI

    PubMed Central

    Pfeiler, Susanne; Khandagale, Avinash B.; Magenau, Astrid; Nichols, Maryana; Heijnen, Harry F. G.; Rinninger, Franz; Ziegler, Tilman; Seveau, Stephanie; Schubert, Sören; Zahler, Stefan; Verschoor, Admar; Latz, Eicke; Massberg, Steffen; Gaus, Katharina; Engelmann, Bernd

    2016-01-01

    The mechanisms protecting from immunopathology during acute bacterial infections are incompletely known. We found that in response to apoptotic immune cells and live or dead Listeria monocytogenes scavenger receptor BI (SR-BI), an anti-atherogenic lipid exchange mediator, activated internalization mechanisms with characteristics of macropinocytosis and, assisted by Golgi fragmentation, initiated autophagic responses. This was supported by scavenger receptor-induced local increases in membrane cholesterol concentrations which generated lipid domains particularly in cell extensions and the Golgi. SR-BI was a key driver of beclin-1-dependent autophagy during acute bacterial infection of the liver and spleen. Autophagy regulated tissue infiltration of neutrophils, suppressed accumulation of Ly6C+ (inflammatory) macrophages, and prevented hepatocyte necrosis in the core of infectious foci. Perifocal levels of Ly6C+ macrophages and Ly6C− macrophages were unaffected, indicating predominant regulation of the focus core. SR-BI-triggered autophagy promoted co-elimination of apoptotic immune cells and dead bacteria but barely influenced bacterial sequestration and survival or inflammasome activation, thus exclusively counteracting damage inflicted by immune responses. Hence, SR-BI- and autophagy promote a surveillance pathway that partially responds to products of antimicrobial defenses and selectively prevents immunity-induced damage during acute infection. Our findings suggest that control of infection-associated immunopathology can be based on a unified defense operation. PMID:27694929

  14. Human apolipoprotein A-I is associated with dengue virus and enhances virus infection through SR-BI.

    PubMed

    Li, Yujia; Kakinami, Cherie; Li, Qi; Yang, Baojun; Li, Hongwei

    2013-01-01

    Diseases caused by dengue virus (DV) infection vary in severity, with symptoms ranging from mild fever to life threatening dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). Clinical studies have shown that significant decrease in the level of lipoproteins is correlated with severe illness in DHF/DSS patients. Available evidence also indicates that lipoproteins including high-density lipoprotein (HDL) and low-density lipoprotein (LDL) are able to facilitate cell entry of HCV or other flaviviruses via corresponding lipoprotein receptors. In this study, we found that pre-incubation of DV with human serum leads to an enhanced DV infectivity in various types of cells. Such enhancement could be due to interactions between serum components and DV particles. Through co-immunoprecipitation we revealed that apolipoprotein A-I (ApoA-I), the major protein component in HDL, is associated with DV particles and is able to promote DV infection. Based on that observation, we further found that siRNA knockdown of the scavenger receptor class B type I (SR-BI), the cell receptor of ApoA-I, abolished the activity of ApoA-I in enhancement of DV infection. This suggests that ApoA-I bridges DV particles and cell receptor SR-BI and facilitates entry of DV into cells. FACS analysis of cell surface dengue antigen after virus absorption further confirmed that ApoA-I enhances DV infection via promoting initial attachment of the virus to cells. These findings illustrate a novel entry route of DV into cells, which may provide insights into the functional importance of lipoproteins in dengue pathogenesis. PMID:23894648

  15. Cholesteryl Ester Transfer Protein Expression Partially Attenuates the Adverse Effects of SR-BI Receptor Deficiency on Cholesterol Metabolism and Atherosclerosis*

    PubMed Central

    El Bouhassani, Majda; Gilibert, Sophie; Moreau, Martine; Saint-Charles, Flora; Tréguier, Morgan; Poti, Francesco; Chapman, M. John; Le Goff, Wilfried; Lesnik, Philippe; Huby, Thierry

    2011-01-01

    Scavenger receptor SR-BI significantly contributes to HDL cholesterol metabolism and atherogenesis in mice. However, the role of SR-BI may not be as pronounced in humans due to cholesteryl ester transfer protein (CETP) activity. To address the impact of CETP expression on the adverse effects associated with SR-BI deficiency, we cross-bred our SR-BI conditional knock-out mouse model with CETP transgenic mice. CETP almost completely restored the abnormal HDL-C distribution in SR-BI-deficient mice. However, it did not normalize the elevated plasma free to total cholesterol ratio characteristic of hepatic SR-BI deficiency. Red blood cell and platelet count abnormalities observed in mice liver deficient for SR-BI were partially restored by CETP, but the elevated erythrocyte cholesterol to phopholipid ratio remained unchanged. Complete deletion of SR-BI was associated with diminished adrenal cholesterol stores, whereas hepatic SR-BI deficiency resulted in a significant increase in adrenal gland cholesterol content. In both mouse models, CETP had no impact on adrenal cholesterol metabolism. In diet-induced atherosclerosis studies, hepatic SR-BI deficiency accelerated aortic lipid lesion formation in both CETP-expressing (4-fold) and non-CETP-expressing (8-fold) mice when compared with controls. Impaired macrophage to feces reverse cholesterol transport in mice deficient for SR-BI in liver, which was not corrected by CETP, most likely contributed by such an increase in atherosclerosis susceptibility. Finally, comparison of the atherosclerosis burden in SR-BI liver-deficient and fully deficient mice demonstrated that SR-BI exerted an atheroprotective activity in extra-hepatic tissues whether CETP was present or not. These findings support the contention that the SR-BI pathway contributes in unique ways to cholesterol metabolism and atherosclerosis susceptibility even in the presence of CETP. PMID:21454568

  16. 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.

  17. 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

  18. 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

  19. 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

    2016-08-18

    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.

  20. 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

  1. Visible-light-mediated Sr-Bi2O3 photocatalysis of tetracycline: kinetics, mechanisms and toxicity assessment.

    PubMed

    Niu, Junfeng; Ding, Shiyuan; Zhang, Liwen; Zhao, Jinbo; Feng, Chenghong

    2013-09-01

    Photodegradation of tetracycline (TC) was investigated in aqueous solution by visible-light-driven photocatalyst Sr-doped β-Bi2O3 (Sr-Bi2O3) prepared via solvothermal synthesis. The decomposition of TC by Sr-Bi2O3 under visible light (λ>420nm) irradiation followed pseudo-first-order kinetics, and the removal ratio reached 91.2% after 120min of irradiation. Sr-Bi2O3 photocatalysis is able to break the naphthol ring of TC which decomposes to m-cresol via dislodging hydroxyl group step by step by photogenerated electron. This mechanism was verified by electron spin resonance measurement, the addition of radical scavengers and the intermediate product analysis, indicating that the photogenerated electron acts as a reductant and can be the key to the degradation process. In contrast, in TiO2 photocatalysis the naphthol ring is broken via oxidation by hydroxyl radical, while in direct photolysis the ring remains intact. In addition, the toxicity of photodegradation products was analyzed by bioluminescence inhibition. After 120min of irradiation by Sr-Bi2O3, the toxicity decreases by 90.6%, which is more substantial than direct photolysis (70%) and TiO2 photocatalysis (80%), indicating that the Sr-Bi2O3 photocatalysis is more eco-friendly than the other two methods.

  2. 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.

  3. [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

  4. 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

  5. Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis.

    PubMed

    Fedashchin, Andrij; Cernota, William H; Gonzalez, Melissa C; Leach, Benjamin I; Kwan, Noelle; Wesley, Roy K; Weber, J Mark

    2015-11-01

    A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ~3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering. PMID:26468041

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. Functional mutagenesis screens reveal the 'cap structure' formation in disulfide-bridge free TASK channels.

    PubMed

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

    2016-01-22

    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.

  14. Atomic Structure and Mutagenesis of T6SS Reveals Interlaced Array Essential to Function

    PubMed Central

    Lee, Bai-Yu; Horwitz, Marcus A.; Zhou, Z. Hong

    2015-01-01

    Summary Type VI secretion systems (T6SSs) are newly identified contractile nanomachines that translocate effector proteins across bacterial membranes. The Francisella pathogenicity island, required for bacterial phagosome escape, intracellular replication and virulence, was presumed to encode a T6SS-like apparatus. Here, we experimentally confirm the identity of this T6SS and, by cryo electron microscopy (cryoEM), show the structure of its post-contraction sheath at 3.7 Å resolution. We demonstrate the assembly of this T6SS by IglA/IglB and secretion of its putative effector proteins in response to environmental stimuli. The sheath has a quaternary structure with handedness opposite that of contracted sheath of T4 phage tail and is organized in an interlaced two-dimensional array by means of β sheet augmentation. By structure-based mutagenesis, we show that this interlacing is essential to secretion, phagosomal escape, and intracellular replication. Our atomic model of the T6SS will facilitate design of drugs targeting this highly prevalent secretion apparatus. PMID:25723168

  15. 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

  16. 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

  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. Ubiquinone-binding site mutagenesis reveals the role of mitochondrial complex II in cell death initiation.

    PubMed

    Kluckova, K; Sticha, M; Cerny, J; Mracek, T; Dong, L; Drahota, Z; Gottlieb, E; Neuzil, J; Rohlena, J

    2015-01-01

    Respiratory complex II (CII, succinate dehydrogenase, SDH) inhibition can induce cell death, but the mechanistic details need clarification. To elucidate the role of reactive oxygen species (ROS) formation upon the ubiquinone-binding (Qp) site blockade, we substituted CII subunit C (SDHC) residues lining the Qp site by site-directed mutagenesis. Cell lines carrying these mutations were characterized on the bases of CII activity and exposed to Qp site inhibitors MitoVES, thenoyltrifluoroacetone (TTFA) and Atpenin A5. We found that I56F and S68A SDHC variants, which support succinate-mediated respiration and maintain low intracellular succinate, were less efficiently inhibited by MitoVES than the wild-type (WT) variant. Importantly, associated ROS generation and cell death induction was also impaired, and cell death in the WT cells was malonate and catalase sensitive. In contrast, the S68A variant was much more susceptible to TTFA inhibition than the I56F variant or the WT CII, which was again reflected by enhanced ROS formation and increased malonate- and catalase-sensitive cell death induction. The R72C variant that accumulates intracellular succinate due to compromised CII activity was resistant to MitoVES and TTFA treatment and did not increase ROS, even though TTFA efficiently generated ROS at low succinate in mitochondria isolated from R72C cells. Similarly, the high-affinity Qp site inhibitor Atpenin A5 rapidly increased intracellular succinate in WT cells but did not induce ROS or cell death, unlike MitoVES and TTFA that upregulated succinate only moderately. These results demonstrate that cell death initiation upon CII inhibition depends on ROS and that the extent of cell death correlates with the potency of inhibition at the Qp site unless intracellular succinate is high. In addition, this validates the Qp site of CII as a target for cell death induction with relevance to cancer therapy. PMID:25950479

  19. Ubiquinone-binding site mutagenesis reveals the role of mitochondrial complex II in cell death initiation

    PubMed Central

    Kluckova, K; Sticha, M; Cerny, J; Mracek, T; Dong, L; Drahota, Z; Gottlieb, E; Neuzil, J; Rohlena, J

    2015-01-01

    Respiratory complex II (CII, succinate dehydrogenase, SDH) inhibition can induce cell death, but the mechanistic details need clarification. To elucidate the role of reactive oxygen species (ROS) formation upon the ubiquinone-binding (Qp) site blockade, we substituted CII subunit C (SDHC) residues lining the Qp site by site-directed mutagenesis. Cell lines carrying these mutations were characterized on the bases of CII activity and exposed to Qp site inhibitors MitoVES, thenoyltrifluoroacetone (TTFA) and Atpenin A5. We found that I56F and S68A SDHC variants, which support succinate-mediated respiration and maintain low intracellular succinate, were less efficiently inhibited by MitoVES than the wild-type (WT) variant. Importantly, associated ROS generation and cell death induction was also impaired, and cell death in the WT cells was malonate and catalase sensitive. In contrast, the S68A variant was much more susceptible to TTFA inhibition than the I56F variant or the WT CII, which was again reflected by enhanced ROS formation and increased malonate- and catalase-sensitive cell death induction. The R72C variant that accumulates intracellular succinate due to compromised CII activity was resistant to MitoVES and TTFA treatment and did not increase ROS, even though TTFA efficiently generated ROS at low succinate in mitochondria isolated from R72C cells. Similarly, the high-affinity Qp site inhibitor Atpenin A5 rapidly increased intracellular succinate in WT cells but did not induce ROS or cell death, unlike MitoVES and TTFA that upregulated succinate only moderately. These results demonstrate that cell death initiation upon CII inhibition depends on ROS and that the extent of cell death correlates with the potency of inhibition at the Qp site unless intracellular succinate is high. In addition, this validates the Qp site of CII as a target for cell death induction with relevance to cancer therapy. PMID:25950479

  20. 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

  1. Mutagenesis of tGCN5 core region reveals two critical surface residues F90 and R140

    SciTech Connect

    Mehta, Kinjal Rajesh; Chan, Yan M.; Lee, Man X.; Yang, Ching Yao; Voloshchuk, Natalya; Montclare, Jin Kim

    2010-09-24

    Research highlights: {yields} Mutagenesis of the tGCN5 core region reveals two residues important for function. {yields} Developed a fluorescent lysate-based activity assay to assess mutants. {yields} Surface-exposed residues F90 and R140 of tGCN5 are critical for H3 acetylation. -- Abstract: Tetrahymena General Control Non-Derepressor 5 (tGCN5) is a critical regulator of gene transcription via acetylation of histones. Since the acetylation ability has been attributed to the 'core region', we perform mutagenesis of residues within the tGCN5 'core region' in order to identify those critical for function and stability. Residues that do not participate in catalysis are identified, mutated and characterized for activity, structure and thermodynamic stability. Variants I107V, Q114L, A121T and A130S maintain the acetylation function relative to wild-type tGCN5, while variants F90Y, F112R and R140H completely abolish function. Of the three non-functional variants, since F112 is mutated into a non-homologous charged residue, a loss in function is expected. However, the remaining two variants are mutated into homologous residues, suggesting that F90 and R140 are critical for the activity of tGCN5. While mutation to homologous residue maintains acetylation of histone H3 for the majority of the variants, the two surface-exposed residues, F90 and R140, appear to be essential for tGCN5 function, structure or stability.

  2. 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.

  3. 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.

  4. Activating Mutations of the TRPML1 Channel Revealed by Proline-scanning Mutagenesis*

    PubMed Central

    Dong, Xian-ping; Wang, Xiang; Shen, Dongbiao; Chen, Su; Liu, Meiling; Wang, Yanbin; Mills, Eric; Cheng, Xiping; Delling, Markus; Xu, Haoxing

    2009-01-01

    The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1V432P) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1V432P-mediated current results from an increased surface expression (trafficking), elevated channel activity (gating), or both. Here we performed systemic Pro substitutions in a region previously implicated in the gating of various 6 transmembrane cation channels. We found that several Pro substitutions displayed gain-of-function (GOF) constitutive activities at both the plasma membrane (PM) and endolysosomal membranes. Although wild-type TRPML1 and non-GOF Pro substitutions localized exclusively in LEL and were barely detectable in the PM, the GOF mutations with high constitutive activities were not restricted to LEL compartments, and most significantly, exhibited significant surface expression. Because lysosomal exocytosis is Ca2+-dependent, constitutive Ca2+ permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca2+ release, hence the surface expression and whole cell current of TRPML1. Indeed, surface staining of lysosome-associated membrane protein-1 (Lamp-1) was dramatically increased in cells expressing GOF TRPML1 channels. We conclude that TRPML1 is an inwardly rectifying, proton-impermeable, Ca2+ and Fe2+/Mn2+ dually permeable cation channel that may be gated by unidentified cellular mechanisms through a conformational change in the cytoplasmic face of the transmembrane 5 (TM5). Furthermore, activation of TRPML1 in LEL may lead to the appearance of TRPML

  5. Effect of oxidizing atmosphere on the electrical properties of SrBi 4Ti 4O 15 thin films obtained by the polymeric precursor method

    NASA Astrophysics Data System (ADS)

    Simões, A. Z.; Ramírez, M. A.; Riccardi, C. S.; Longo, E.; Varela, J. A.

    2008-12-01

    Strontium bismuth titanate (SrBi 4Ti 4O 15) thin films were deposited on (111) Pt/Ti/SiO 2/Si substrates by spin coating from the polymeric precursor method. Annealing in static air and dynamic oxygen atmosphere was performed at 700 °C for 2 h. The films were characterized by X-ray diffraction, atomic force microscopy and electric properties. The dielectric properties of SrBi 4Ti 4O 15 films were found to be remarkably sensitive to the annealing atmosphere. The C- V characteristics of the metal-ferroelectric metal structure showed a typical butterfly loop that confirms the ferroelectric properties of the film related to the domains switching. SrBi 4Ti 4O 15 thin films annealed in oxygen atmosphere showed lower ferroelectric behavior indicating a weak ferroelectricity along c-axis direction.

  6. 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

  7. 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

  8. 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.

  9. CRISPR/Cas9 Mutagenesis Reveals Versatile Roles of Hox Genes in Crustacean Limb Specification and Evolution.

    PubMed

    Martin, Arnaud; Serano, Julia M; Jarvis, Erin; Bruce, Heather S; Wang, Jennifer; Ray, Shagnik; Barker, Carryn A; O'Connell, Liam C; Patel, Nipam H

    2016-01-11

    Crustaceans possess a diverse array of specialized limbs. Although shifts in Hox gene expression domains have been postulated to play a role in generating this limb diversity, little functional data have been provided to understand the precise roles of Hox genes during crustacean development. We used a combination of CRISPR/Cas9-targeted mutagenesis and RNAi knockdown to decipher the function of the six Hox genes expressed in the developing mouth and trunk of the amphipod Parhyale hawaiensis. These experimentally manipulated animals display specific and striking homeotic transformations. We found that abdominal-A (abd-A) and Abdominal-B (Abd-B) are required for proper posterior patterning, with knockout of Abd-B resulting in an animal with thoracic type legs along what would have been an abdomen, and abd-A disruption generating a simplified body plan characterized by a loss of specialization in both abdominal and thoracic appendages. In the thorax, Ubx is necessary for gill development and for repression of gnathal fate, and Antp dictates claw morphology. In the mouth, Scr and Antp confer the part-gnathal, part-thoracic hybrid identity of the maxilliped, and Scr and Dfd prevent antennal identity in posterior head segments. Our results allow us to define the role Hox genes play in specifying each appendage type in Parhyale, including the modular nature by which some appendages are patterned by Hox gene inputs. In addition, we define how changes in Hox gene expression have generated morphological differences between crustacean species. Finally, we also highlight the utility of CRISPR/Cas9-based somatic mutagenesis in emerging model organisms.

  10. Transposon mutagenesis reveals cooperation of ETS family transcription factors with signaling pathways in erythro-megakaryocytic leukemia

    PubMed Central

    Tang, Jian Zhong; Carmichael, Catherine L.; Shi, Wei; Metcalf, Donald; Ng, Ashley P.; Hyland, Craig D.; Jenkins, Nancy A.; Copeland, Neal G.; Howell, Viive M.; Zhao, Zhizhuang Joe; Smyth, Gordon K.; Kile, Benjamin T.; Alexander, Warren S.

    2013-01-01

    To define genetic lesions driving leukemia, we targeted cre-dependent Sleeping Beauty (SB) transposon mutagenesis to the blood-forming system using a hematopoietic-selective vav 1 oncogene (vav1) promoter. Leukemias of diverse lineages ensued, most commonly lymphoid leukemia and erythroleukemia. The inclusion of a transgenic allele of Janus kinase 2 (JAK2)V617F resulted in acceleration of transposon-driven disease and strong selection for erythroleukemic pathology with transformation of bipotential erythro-megakaryocytic cells. The genes encoding the E-twenty-six (ETS) transcription factors Ets related gene (Erg) and Ets1 were the most common sites for transposon insertion in SB-induced JAK2V617F-positive erythroleukemias, present in 87.5% and 65%, respectively, of independent leukemias examined. The role of activated Erg was validated by reproducing erythroleukemic pathology in mice transplanted with fetal liver cells expressing translocated in liposarcoma (TLS)-ERG, an activated form of ERG found in human leukemia. Via application of SB mutagenesis to TLS-ERG–induced erythroid transformation, we identified multiple loci as likely collaborators with activation of Erg. Jak2 was identified as a common transposon insertion site in TLS-ERG–induced disease, strongly validating the cooperation between JAK2V617F and transposon insertion at the Erg locus in the JAK2V617F-positive leukemias. Moreover, loci expressing other regulators of signal transduction pathways were conspicuous among the common transposon insertion sites in TLS-ERG–driven leukemia, suggesting that a key mechanism in erythroleukemia may be the collaboration of lesions disturbing erythroid maturation, most notably in genes of the ETS family, with mutations that reduce dependence on exogenous signals. PMID:23533276

  11. 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

  12. 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

  13. Atomic vapour deposition (AVD) of SrBi 2Ta 2O 9 using an all alkoxide precursor

    NASA Astrophysics Data System (ADS)

    Chalker, Paul R.; Potter, Richard J.; Roberts, John L.; Jones, Anthony C.; Smith, Lesley M.; Schumacher, Marcus

    2004-12-01

    A "single-source" Sr-Ta heterometal alkoxide precursor, Sr[Ta(OEt) 5(dmae)] 2 (dmae=OCH 2CH 2NMe 2), has been used for atomic vapour deposition (AVD) of SrBi 2Ta 2O 9 (SBT). This single-source precursor is designed to alleviate the mismatch between conventional Sr and Ta sources. Strontium tantalate thin films were deposited on silicon using the Sr[Ta(OEt) 5(dmae)] 2, and the optimum temperatures for deposition of strontium tantalate with a Sr:Ta ratio of 0.5 was found to be ˜510 °C. Deposition of Bi-oxide films using Bi(mmp) 3 (mmp=OCMe 2CH 2OMe) indicates similar decomposition behaviour to the Sr-Ta alkoxide precursor, demonstrating its suitability as a complementary source of Bi for SBT. The co-incorporation of Bi and Sr within the SBT films is promoted through the deposition of bismuth oxide/strontium tantalate super lattices. After post-growth annealing the super lattices are converted to strontium bismuth tantalate thin films.

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

    PubMed Central

    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.

    2015-01-01

    Summary 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

  15. 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.

  16. Electrostatic interaction between oxysterol-binding protein and VAMP-associated protein A revealed by NMR and mutagenesis studies.

    PubMed

    Furuita, Kyoko; Jee, JunGoo; Fukada, Harumi; Mishima, Masaki; Kojima, Chojiro

    2010-04-23

    Oxysterol-binding protein (OSBP), a cytosolic receptor of cholesterol and oxysterols, is recruited to the endoplasmic reticulum by binding to the cytoplasmic major sperm protein (MSP) domain of integral endoplasmic reticulum protein VAMP-associated protein-A (VAP-A), a process essential for the stimulation of sphingomyelin synthesis by 25-hydroxycholesterol. To delineate the interaction mechanism between VAP-A and OSBP, we determined the complex structure between the VAP-A MSP domain (VAP-A(MSP)) and the OSBP fragment containing a VAP-A binding motif FFAT (OSBP(F)) by NMR. This solution structure explained that five of six conserved residues in the FFAT motif are required for the stable complex formation, and three of five, including three critical intermolecular electrostatic interactions, were not explained before. By combining NMR relaxation and titration, isothermal titration calorimetry, and mutagenesis experiments with structural information, we further elucidated the detailed roles of the FFAT motif and underlying motions of VAP-A(MSP), OSBP(F), and the complex. Our results show that OSBP(F) is disordered in the free state, and VAP-A(MSP) and OSBP(F) form a final complex by means of intermediates, where electrostatic interactions through acidic residues, including an acid patch preceding the FFAT motif, probably play a collective role. Additionally, we report that the mutation that causes the familial motor neuron disease decreases the stability of the MSP domain. PMID:20178991

  17. 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

  18. 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

  19. 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

  20. Site-directed mutagenesis of a tetrameric dandelion polyphenol oxidase (PPO-6) reveals the site of subunit interaction.

    PubMed

    Dirks-Hofmeister, Mareike E; Inlow, Jennifer K; Moerschbacher, Bruno M

    2012-09-01

    Polyphenol oxidases (PPOs) catalyze the oxidation of ortho-diphenols to the corresponding quinones (EC 1.10.3.1). In plants PPOs appear in gene families, and the corresponding isoenzymes are located to the thylakoid lumen of chloroplasts. Although plant PPOs are often discussed with regard to their role in defense reactions, a common physiological function has not yet been defined. We analyzed a tetrameric PPO isoenzyme (PPO-6) from dandelion (Taraxacum officinale) heterologously expressed in Escherichia coli, and found it to display cooperativity in catalysis, a phenomenon that has rarely been shown for plant PPOs previously. The identification of a surface-exposed cysteine (197) through molecular modeling followed by site-directed mutagenesis proved this amino acid residue to stabilize the tetramer via a disulfide linkage. The C197S-mutein still forms a tetrameric structure but shows impaired enzymatic efficiency and cooperativity and a reduction in stability. These findings indicate that oligomerization may be a physiological requirement for PPO-6 stability and function in vivo and raise new questions regarding distinct functions for specific PPO isoenzymes in plants.

  1. Electrostatic interaction between oxysterol-binding protein and VAMP-associated protein A revealed by NMR and mutagenesis studies.

    PubMed

    Furuita, Kyoko; Jee, JunGoo; Fukada, Harumi; Mishima, Masaki; Kojima, Chojiro

    2010-04-23

    Oxysterol-binding protein (OSBP), a cytosolic receptor of cholesterol and oxysterols, is recruited to the endoplasmic reticulum by binding to the cytoplasmic major sperm protein (MSP) domain of integral endoplasmic reticulum protein VAMP-associated protein-A (VAP-A), a process essential for the stimulation of sphingomyelin synthesis by 25-hydroxycholesterol. To delineate the interaction mechanism between VAP-A and OSBP, we determined the complex structure between the VAP-A MSP domain (VAP-A(MSP)) and the OSBP fragment containing a VAP-A binding motif FFAT (OSBP(F)) by NMR. This solution structure explained that five of six conserved residues in the FFAT motif are required for the stable complex formation, and three of five, including three critical intermolecular electrostatic interactions, were not explained before. By combining NMR relaxation and titration, isothermal titration calorimetry, and mutagenesis experiments with structural information, we further elucidated the detailed roles of the FFAT motif and underlying motions of VAP-A(MSP), OSBP(F), and the complex. Our results show that OSBP(F) is disordered in the free state, and VAP-A(MSP) and OSBP(F) form a final complex by means of intermediates, where electrostatic interactions through acidic residues, including an acid patch preceding the FFAT motif, probably play a collective role. Additionally, we report that the mutation that causes the familial motor neuron disease decreases the stability of the MSP domain.

  2. Electrostatic Interaction between Oxysterol-binding Protein and VAMP-associated Protein A Revealed by NMR and Mutagenesis Studies*

    PubMed Central

    Furuita, Kyoko; Jee, JunGoo; Fukada, Harumi; Mishima, Masaki; Kojima, Chojiro

    2010-01-01

    Oxysterol-binding protein (OSBP), a cytosolic receptor of cholesterol and oxysterols, is recruited to the endoplasmic reticulum by binding to the cytoplasmic major sperm protein (MSP) domain of integral endoplasmic reticulum protein VAMP-associated protein-A (VAP-A), a process essential for the stimulation of sphingomyelin synthesis by 25-hydroxycholesterol. To delineate the interaction mechanism between VAP-A and OSBP, we determined the complex structure between the VAP-A MSP domain (VAP-AMSP) and the OSBP fragment containing a VAP-A binding motif FFAT (OSBPF) by NMR. This solution structure explained that five of six conserved residues in the FFAT motif are required for the stable complex formation, and three of five, including three critical intermolecular electrostatic interactions, were not explained before. By combining NMR relaxation and titration, isothermal titration calorimetry, and mutagenesis experiments with structural information, we further elucidated the detailed roles of the FFAT motif and underlying motions of VAP-AMSP, OSBPF, and the complex. Our results show that OSBPF is disordered in the free state, and VAP-AMSP and OSBPF form a final complex by means of intermediates, where electrostatic interactions through acidic residues, including an acid patch preceding the FFAT motif, probably play a collective role. Additionally, we report that the mutation that causes the familial motor neuron disease decreases the stability of the MSP domain. PMID:20178991

  3. Mutagenesis of Zinc Ligand Residue Cys221 Reveals Plasticity in the IMP-1 Metallo-β-Lactamase Active Site

    PubMed Central

    Horton, Lori B.; Shanker, Sreejesh; Mikulski, Rose; Brown, Nicholas G.; Phillips, Kevin J.; Lykissa, Ernest; Venkataram Prasad, B. V.

    2012-01-01

    Metallo-β-lactamases catalyze the hydrolysis of a broad range of β-lactam antibiotics and are a concern for the spread of drug resistance. To analyze the determinants of enzyme structure and function, the sequence requirements for the subclass B1 IMP-1 β-lactamase zinc binding residue Cys221 were tested by saturation mutagenesis and evaluated for protein expression, as well as hydrolysis of β-lactam substrates. The results indicated that most substitutions at position 221 destabilized the enzyme. Only the enzymes containing C221D and C221G substitutions were expressed well in Escherichia coli and exhibited catalytic activity toward β-lactam antibiotics. Despite the lack of a metal-chelating group at position 221, the C221G enzyme exhibited high levels of catalytic activity in the presence of exogenous zinc. Molecular modeling suggests the glycine substitution is unique among substitutions in that the complete removal of the cysteine side chain allows space for a water molecule to replace the thiol and coordinate zinc at the Zn2 zinc binding site to restore function. Multiple methods were used to estimate the C221G Zn2 binding constant to be 17 to 43 μM. Studies of enzyme function in vivo in E. coli grown on minimal medium showed that both IMP-1 and the C221G mutant exhibited compromised activity when zinc availability was low. Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable. PMID:22908171

  4. In vitro and in vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B type I (SR-BI) to the PDZ1 Domain of its Cytoplasmic Adaptor Protein PDZK1

    SciTech Connect

    O Kocher; G Birrane; K Tsukamoto; S Fenske; A Yesilaltay; R Pal; K Daniels; J Ladias; M Krieger

    2011-12-31

    The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys(14)-Xaa(4)-Asn(19)-Tyr-Gly-Phe-Phe-Leu(24)), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI ((503)VLQEAKL(509)). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (K(d)) of the K14A and F22A mutants were 3.2 and 4.0 ?M, respectively, similar to 2.6 ?M measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI ((505)QEAKL(509)) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10-20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.

  5. Phaleria macrocarpa Boerl. (Thymelaeaceae) leaves increase SR-BI expression and reduce cholesterol levels in rats fed a high cholesterol diet.

    PubMed

    Andriani, Yosie; Tengku-Muhammad, Tengku Sifzizul; Mohamad, Habsah; Saidin, Jasnizat; Syamsumir, Desy Fitrya; Chew, Guat-Siew; Abdul Wahid, Mohd Effendy

    2015-01-01

    In vitro and in vivo studies of the activity of Phaleria macrocarpa Boerl (Thymelaeaceae) leaves against the therapeutic target for hypercholesterolemia were done using the HDL receptor (SR-BI) and hypercholesterolemia-induced Sprague Dawley rats. The in vitro study showed that the active fraction (CF6) obtained from the ethyl acetate extract (EMD) and its component 2',6',4-trihydroxy-4'-methoxybenzophenone increased the SR-BI expression by 95% and 60%, respectively. The in vivo study has proven the effect of EMD at 0.5 g/kgbw dosage in reducing the total cholesterol level by 224.9% and increasing the HDL cholesterol level by 157% compared to the cholesterol group. In the toxicity study, serum glutamate oxalate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) activity were observed to be at normal levels. The liver histology also proved no toxicity and abnormalities in any of the treatment groups, so it can be categorized as non-toxic to the rat liver. The findings taken together show that P. macrocarpa leaves are safe and suitable as an alternative control and prevention treatment for hypercholesterolemia in Sprague Dawley rats. PMID:25759957

  6. Benzo-fused lactams from a diversity-oriented synthesis (DOS) library as inhibitors of scavenger receptor BI (SR-BI)-mediated lipid uptake.

    PubMed

    Dockendorff, Chris; Faloon, Patrick W; Pu, Jun; Yu, Miao; Johnston, Stephen; Bennion, Melissa; Penman, Marsha; Nieland, Thomas J F; Dandapani, Sivaraman; Perez, José R; Munoz, Benito; Palmer, Michelle A; Schreiber, Stuart L; Krieger, Monty

    2015-01-01

    We report a new series of 8-membered benzo-fused lactams that inhibit cellular lipid uptake from HDL particles mediated by Scavenger Receptor, Class B, Type I (SR-BI). The series was identified via a high-throughput screen of the National Institutes of Health Molecular Libraries Small Molecule Repository (NIH MLSMR), measuring the transfer of the fluorescent lipid DiI from HDL particles to CHO cells overexpressing SR-BI. The series is part of a previously reported diversity-oriented synthesis (DOS) library prepared via a build-couple-pair approach. Detailed structure-activity relationship (SAR) studies were performed with a selection of the original library, as well as additional analogs prepared via solution phase synthesis. These studies demonstrate that the orientation of the substituents on the aliphatic ring have a critical effect on activity. Additionally, a lipophilic group is required at the western end of the molecule, and a northern hydroxyl group and a southern sulfonamide substituent also proved to be optimal. Compound 2p was found to possess a superior combination of potency (av IC50=0.10μM) and solubility (79μM in PBS), and it was designated as probe ML312. PMID:25900219

  7. Orientation of a key glutamine residue in the BLUF domain from AppA revealed by mutagenesis, spectroscopy, and quantum chemical calculations.

    PubMed

    Unno, Masashi; Masuda, Shinji; Ono, Taka-aki; Yamauchi, Seigo

    2006-05-01

    The flavin-adenine-dinucleotide-binding BLUF domain constitutes a new class of blue-light receptors, and the N-terminal domain of AppA is a representative of this family. A crystal structure of the BLUF domain from AppA suggested that a conserved Gln63 forms a hydrogen bond with the flavin N5 atom. Upon light excitation, this residue is proposed to undergo a approximately 180 degrees rotation that leads to a rearrangement of a hydrogen bonding network. However, crystallographic studies on the other BLUF proteins claimed an opposite orientation for the glutamine residue. In this communication, we have revealed the presence of a Gln63-to-N5 hydrogen bond in the dark state of AppA by a combined approach of mutagenesis, spectroscopy, and quantum chemical calculations. The present finding supports the view that the reorientation of the Gln63 side chain is a key event in the signaling state formation of BLUF proteins.

  8. Newly identified essential amino acid residues affecting Δ8-sphingolipid desaturase activity revealed by site-directed mutagenesis.

    PubMed

    Li, Shu-Fen; Song, Li-Ying; Zhang, Guo-Jun; Yin, Wei-Bo; Chen, Yu-Hong; Wang, Richard R-C; Hu, Zan-Min

    2011-12-01

    In order to identify amino acid residues crucial for the enzymatic activity of Δ(8)-sphingolipid desaturases, a sequence comparison was performed among Δ(8)-sphingolipid desaturases and Δ(6)-fatty acid desaturases from various plants. In addition to the known conserved cytb(5) (cytochrome b(5)) HPGG motif and three conserved histidine boxes, they share additional 15 completely conserved residues. A series of site-directed mutants were generated using our previously isolated Δ(8)-sphingolipid desaturase gene from Brassica rapa to evaluate the importance of these residues to the enzyme function. The mutants were functionally characterized by heterologous expression in yeast, allowing the identification of the products of the enzymes. The results revealed that residues H63, N203, D208, D210, and G368 were obligatorily required for the enzymatic activity, and substitution of the residues F59, W190, W345, L369 and Q372 markedly decreased the enzyme activity. Among them, replacement of the residues W190, L369 and Q372 also has significant influence on the ratio of the two enzyme products. Information obtained in this work provides the molecular basis for the Δ(8)-sphingolipid desaturase activity and aids in our understanding of the structure-function relationships of the membrane-bound desaturases.

  9. Site-directed mutagenesis of HgcA and HgcB reveals amino acid residues important for mercury methylation.

    PubMed

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

    2015-05-01

    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.

  10. CryoEM and mutagenesis reveal that the smallest capsid protein cements and stabilizes Kaposi's sarcoma-associated herpesvirus capsid.

    PubMed

    Dai, Xinghong; Gong, Danyang; Xiao, Yuchen; Wu, Ting-Ting; Sun, Ren; Zhou, Z Hong

    2015-02-17

    With just one eighth the size of the major capsid protein (MCP), the smallest capsid protein (SCP) of human tumor herpesviruses--Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV)--is vital to capsid assembly, yet its mechanism of action is unknown. Here, by cryoEM of KSHV at 6-Å resolution, we show that SCP forms a crown on each hexon and uses a kinked helix to cross-link neighboring MCP subunits. SCP-null mutation decreased viral titer by 1,000 times and impaired but did not fully abolish capsid assembly, indicating an important but nonessential role of SCP. By truncating the C-terminal half of SCP and performing cryoEM reconstruction, we demonstrate that SCP's N-terminal half is responsible for the observed structure and function whereas the C-terminal half is flexible and dispensable. Serial truncations further highlight the critical importance of the N-terminal 10 aa, and cryoEM reconstruction of the one with six residues truncated localizes the N terminus of SCP in the cryoEM density map and enables us to construct a pseudoatomic model of SCP. Fitting of this SCP model and a homology model for the MCP upper domain into the cryoEM map reveals that SCP binds MCP largely via hydrophobic interactions and the kinked helix of SCP bridges over neighboring MCPs to form noncovalent cross-links. These data support a mechanistic model that tumor herpesvirus SCP reinforces the capsid for genome packaging, thus acting as a cementing protein similar to those found in many bacteriophages.

  11. 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

  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. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. Growth Behavior of c-Axis-Oriented Epitaxial SrBi2Ta2O9 Films on SrTiO3 Substrates with Atomic Scale Step Structure

    NASA Astrophysics Data System (ADS)

    Takahashi, Kenji; Suzuki, Muneyasu; Yoshimoto, Mamoru; Funakubo, Hiroshi

    2006-02-01

    c-Axis-oriented ultra thin SrBi2Ta2O9 films were epitaxially grown on atomically flat (100)SrTiO3 substrates by pulse-gas-introduced metalorganic chemical vapor deposition, and their growth behavior was observed by atomic force microscopy (AFM). Growth-time-resolved AFM images clearly showed that a completely filled SrBi2Ta2O9 layer was laid under an incompletely grown half-unit-cell two-dimensional (2D)-island layer, indicating the Frank-van der Merwe (layer-by-layer) growth mode. This is the first step-by-step direct observation of layer-by-layer growth of c-axis-oriented bismuth layer-structured dielectric (BLD) films and is considered to be the origin of the thickness-independent smooth surface of c-axis-oriented BLD films.

  19. Stacked Pt/SrBi2Ta2-xNbxO9/Pt/IrOx/Ir Capacitor on Poly Plug

    NASA Astrophysics Data System (ADS)

    Kweon, Soon Yong; Choi, Si Kyung; Yang, Woo Seok; Yeom, Seung Jin; Roh, Jae Sung

    2002-01-01

    A Pt/SrBi2Ta2-xNbxO9(SBTN)/Pt/IrOx/Ir capacitor was successfully fabricated up to the stage of metal-1 etching process on a polysilicon plug for mega-bit ferroelectric random access memory. The integration processes include the chemical-mechanical polishing technique, buried TiN barrier structure and electrode technologies for high thermal stability, and a low-temperature process for SBTN film. The thickness of the iridium layer was the most important factor in controlling the contact resistance of the plug. The Pt thickness also affected the contact resistance of the plug. The best contact resistance of the plug was about 2.0 kΩ/plug at the maximum process temperature of 750°C for 3 min in oxygen ambient at the contact size of φ 0.30 μm. Hysteresis curves of the SBTN capacitor were obtained after the metal-1 etching process. The capacitor size dependency of the polarization was not observed in the range of 0.30-25 μm2 and the values of the sensing polarization were about 10 μC/cm2 at the applied voltage of 3 V@. In addition, the capacitor exhibited no fatigue loss up to 5× 1010 cycles at the switching voltage of 3 V.@

  20. Preparation and Characterization of SrBi2(Ta1-xNbx)2O9 Thin Films by Metalorganic Chemical Vapor Deposition from Two Organometallic Source Bottles

    NASA Astrophysics Data System (ADS)

    Mitsuya, Masatoshi; Ishikawa, Katsuyuki; Nukaga, Norimasa; Funakubo, Hiroshi

    2000-06-01

    SrBi2(Ta1-xNbx)2O9 (SBTN) thin films were first prepared on Pt/Ti/SiO2/Si substrates by metalorganic chemical vapor deposition (MOCVD) with high compositional reproducibility. Bi(CH3)3, a mixture of Sr[Ta(O\\cdotC2H5)6]2 and Sr[Nb(O\\cdotC2H5)6]2, and O2 gas were used as sources. The Nb/(Ta+Nb) ratio in the film was almost the same as that of the source materials. The film, deposited at 500°C following heat treatment at 800°C for 30 min in O2 atmosphere, consisted of an almost single phase of SBTN@. The remanent polarization and the coercive field of the 330 nm-thick film were 8.5 μC/cm2 and 91 kV/cm, respectively. This film showed negligible fatigue after 5× 1010 polarization switching cycles.

  1. Synthesis and characterization of nanosized SrBi{sub 2}Ta{sub 2}O{sub 9} powder by a novel sol-gel process

    SciTech Connect

    Wang Wen; Jia Dechang; Zhou Yu; Ye Feng

    2002-12-01

    Chemically homogeneous SrBi{sub 2}Ta{sub 2}O{sub 9} (SBT) sol was synthesized using ethoxy tantalum, strontium acetate, and bismuth subnitrate as starting materials, methoxyethylene as a solvent and acetic acid (HOAc) as a catalyst. Single-phased perovskite phase SBT ferroelectric ultrafine powder was obtained after the dried gel was treated at 350 deg. C for 30 min and calcined at 800 deg. C for 1 h. FT-IR, XRD, TEM and TG-DTA were employed to investigate the transformation processes of sol to gel and gel to ultrafine SBT powder. Acetic acid not only acts as an acid catalyst, but also changes the alkoxide precursor as a ligand at a molecular level. Bidentate acetates replace OR groups and are directly bounded to the tantalum, leading to the formation of Ta (OR){sub x}(OAc){sub 5-x}. The perovskite SBT phase formed via intermediate phase Bi{sub 3}TaO{sub 7} and a Bi-deficient pyrocholore phase.

  2. 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 Central

    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

  3. Large-Scale Transposon Mutagenesis of Photobacterium profundum SS9 Reveals New Genetic Loci Important for Growth at Low Temperature and High Pressure▿

    PubMed Central

    Lauro, Federico M.; Tran, Khiem; Vezzi, Alessandro; Vitulo, Nicola; Valle, Giorgio; Bartlett, Douglas H.

    2008-01-01

    Microorganisms adapted to piezopsychrophilic growth dominate the majority of the biosphere that is at relatively constant low temperatures and high pressures, but the genetic bases for the adaptations are largely unknown. Here we report the use of transposon mutagenesis with the deep-sea bacterium Photobacterium profundum strain SS9 to isolate dozens of mutant strains whose growth is impaired at low temperature and/or whose growth is altered as a function of hydrostatic pressure. In many cases the gene mutation-growth phenotype relationship was verified by complementation analysis. The largest fraction of loci associated with temperature sensitivity were involved in the biosynthesis of the cell envelope, in particular the biosynthesis of extracellular polysaccharide. The largest fraction of loci associated with pressure sensitivity were involved in chromosomal structure and function. Genes for ribosome assembly and function were found to be important for both low-temperature and high-pressure growth. Likewise, both adaptation to temperature and adaptation to pressure were affected by mutations in a number of sensory and regulatory loci, suggesting the importance of signal transduction mechanisms in adaptation to either physical parameter. These analyses were the first global analyses of genes conditionally required for low-temperature or high-pressure growth in a deep-sea microorganism. PMID:18156275

  4. 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

  5. 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

  6. Systematic Targeted Mutagenesis of Brucella melitensis 16M Reveals a Major Role for GntR Regulators in the Control of Virulence

    PubMed Central

    Haine, Valérie; Sinon, Audrey; Van Steen, Frédéric; Rousseau, Stéphanie; Dozot, Marie; Lestrate, Pascal; Lambert, Christophe; Letesson, Jean-Jacques; De Bolle, Xavier

    2005-01-01

    In order to identify transcriptional regulators involved in virulence gene control in Brucella melitensis, we generated a collection of 88 mutants in the AraC, ArsR, Crp, DeoR, GntR, IclR, LysR, MerR, RpiR, and TetR families of regulators. This collection was named LiMuR (library of mutants for regulators). We developed a method to test several mutants simultaneously in one animal in order to identify those unable to survive. This method, called the plasmid-tagged mutagenesis method, was used to test the residual virulence of mutants after 1 week in a mouse model of infection. Ten attenuated mutants, of which six and three belong to the GntR and LysR families, respectively, were identified and individually confirmed to replicate at lower rates in mice. Among these 10 mutants, only gntR10 and arsR6 are attenuated in cellular models. The LiMuR also allows simple screenings to identify regulators of a particular gene or operon. As a first example, we analyzed the expression of the virB operon in the LiMuR mutants. We carried out Western blottings of whole-cell extracts to analyze the production of VirB proteins using polyclonal antisera against VirB proteins. Four mutants produced small amounts of VirB proteins, and one mutant overexpressed VirB proteins compared to the wild-type strain. In these five mutants, reporter analysis using the virB promoter fused to lacZ showed that three mutants control virB at the transcriptional level. The LiMuR is a resource that will provide straightforward identification of regulators involved in the control of genes of interest. PMID:16113274

  7. Tertiary Structural and Functional Analyses of a Viroid RNA Motif by Isostericity Matrix and Mutagenesis Reveal Its Essential Role in Replication

    PubMed Central

    Zhong, Xuehua; Leontis, Neocles; Qian, Shuiming; Itaya, Asuka; Qi, Yijun; Boris-Lawrie, Kathleen; Ding, Biao

    2006-01-01

    RNA-templated RNA replication is essential for viral or viroid infection, as well as for regulation of cellular gene expression. Specific RNA motifs likely regulate various aspects of this replication. Viroids of the Pospiviroidae family, as represented by the Potato spindle tuber viroid (PSTVd), replicate in the nucleus by utilizing DNA-dependent RNA polymerase II. We investigated the role of the loop E (sarcin/ricin) motif of the PSTVd genomic RNA in replication. A tertiary-structural model of this motif, inferred by comparative sequence analysis and comparison with nuclear magnetic resonance and X-ray crystal structures of loop E motifs in other RNAs, is presented in which core non-Watson-Crick base pairs are precisely specified. Isostericity matrix analysis of these base pairs showed that the model accounts for the reported natural sequence variations and viable experimental mutations in loop E motifs of PSTVd and other viroids. Furthermore, isostericity matrix analysis allowed us to design disruptive, as well as compensatory, mutations of PSTVd loop E. Functional analyses of such mutants by in vitro and in vivo experiments demonstrated that loop E structural integrity is crucial for replication, specifically during transcription. Our results suggest that the PSTVd loop E motif exists and functions in vivo and provide loss-of-function genetic evidence for the essential role of a viroid RNA three-dimensional motif in rolling-circle replication. The use of isostericity matrix analysis of non-Watson-Crick base pairing to rationalize mutagenesis of tertiary motifs and systematic in vitro and in vivo functional assays of mutants offers a novel, comprehensive approach to elucidate the tertiary-structure-function relationships for RNA motifs of general biological significance. PMID:16912306

  8. 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.

  9. 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 Central

    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.

    2015-01-01

    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

  10. Mutagenesis of SNM1, Which Encodes a Protein Component of the Yeast RNase MRP, Reveals a Role for This Ribonucleoprotein Endoribonuclease in Plasmid Segregation

    PubMed Central

    Cai, Ti; Reilly, Tracey R.; Cerio, Michael; Schmitt, Mark E.

    1999-01-01

    RNase MRP is a ribonucleoprotein endoribonuclease that has been shown to have roles in both mitochondrial DNA replication and nuclear 5.8S rRNA processing. SNM1 encodes an essential 22.5-kDa protein that is a component of yeast RNase MRP. It is an RNA binding protein that binds the MRP RNA specifically. This 198-amino-acid protein can be divided into three structural regions: a potential leucine zipper near the amino terminus, a binuclear zinc cluster in the middle region, and a serine- and lysine-rich region near the carboxy terminus. We have performed PCR mutagenesis of the SNM1 gene to produce 17 mutants that have a conditional phenotype for growth at different temperatures. Yeast strains carrying any of these mutations as the only copy of snm1 display an rRNA processing defect identical to that in MRP RNA mutants. We have characterized these mutant proteins for RNase MRP function by examining 5.8S rRNA processing, MRP RNA binding in vivo, and the stability of the RNase MRP RNA. The results indicate two separate functional domains of the protein, one responsible for binding the MRP RNA and a second that promotes substrate cleavage. The Snm1 protein appears not to be required for the stability of the MRP RNA, but very low levels of the protein are required for processing of the 5.8S rRNA. Surprisingly, a large number of conditional mutations that resulted from nonsense and frameshift mutations throughout the coding regions were identified. The most severe of these was a frameshift at amino acid 7. These mutations were found to be undergoing translational suppression, resulting in a small amount of full-length Snm1 protein. This small amount of Snm1 protein was sufficient to maintain enough RNase MRP activity to support viability. Translational suppression was accomplished in two ways. First, CEN plasmid missegregation leads to plasmid amplification, which in turn leads to SNM1 mRNA overexpression. Translational suppression of a small amount of the superabundant

  11. Mutagenesis Reveals Structure–Activity Parallels between Human A2A Adenosine Receptors and Biogenic Amine G Protein-Coupled Receptors

    PubMed Central

    Jiang, Qiaoling; Lee, Brian X.; Glashofer, Marc; van Rhee, A. Michiel; Jacobson, Kenneth A.

    2012-01-01

    Structure–affinity relationships for ligand binding at the human A2A adenosine receptor have been probed using site-directed mutagenesis in the transmembrane helical domains (TMs). The mutant receptors were expressed in COS-7 cells and characterized by binding of the radioligands [3H]CGS21680, [3H]NECA, and [3H]XAC. Three residues, at positions essential for ligand binding in other G protein-coupled receptors, were individually mutated. The residue V(3.32) in the A2A receptor that is homologous to the essential aspartate residue of TM3 in the biogenic amine receptors, i.e., V84(3.32), may be substituted with L (present in the A3 receptor) but not with D (in biogenic amine receptors) or A. H250(6.52), homologous to the critical N507 of rat m3 muscarinic acetylcholine receptors, may be substituted with other aromatic residues or with N but not with A (Kim et al. J. Biol. Chem. 1995, 270, 13987–13997). H278(7.43), homologous to the covalent ligand anchor site in rhodopsin, may not be substituted with either A, K, or N. Both V84L(3.32) and H250N(6.52) mutant receptors were highly variable in their effect on ligand competition depending on the structural class of the ligand. Adenosine-5′-uronamide derivatives were more potent at the H250N(6.52) mutant receptor than at wild type receptors. Xanthines tended to be close in potency (H250N(6.52)) or less potent (V84L-(3.32)) than at wild type receptors. The affinity of CGS21680 increased as the pH was lowered to 5.5 in both the wild type and H250N(6.52) mutant receptors. Thus, protonation of H250-(6.52) is not involved in this pH dependence. These data are consistent with a molecular model predicting the proximity of bound agonist ligands to TM3, TM5, TM6, and TM7. PMID:9258366

  12. 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

  13. 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.

  14. 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)

  15. The Mutagenesis Assistant Program.

    PubMed

    Verma, Rajni; Wong, Tuck Seng; Schwaneberg, Ulrich; Roccatano, Danilo

    2014-01-01

    Mutagenesis Assistant Program (MAP) is a web-based statistical tool to develop directed evolution strategies by investigating the consequences at the amino acid level of the mutational biases of random mutagenesis methods on any given gene. The latest development of the program, the MAP(2.0)3D server, correlates the generated amino acid substitution patterns of a specific random mutagenesis method to the sequence and structural information of the target protein. The combined information can be used to select an experimental strategy that improves the chances of obtaining functionally efficient and/or stable enzyme variants. Hence, the MAP(2.0)3D server facilitates the "in silico" prescreening of the target gene by predicting the amino acid diversity generated in a random mutagenesis library. Here, we describe the features of MAP(2.0)3D server by analyzing, as an example, the cytochrome P450BM3 monooxygenase (CYP102A1). The MAP(2.0)3D server is available publicly at http://map.jacobs-university.de/map3d.html.

  16. Site-directed mutagenesis.

    PubMed

    Bachman, Julia

    2013-01-01

    Site-directed mutagenesis is a PCR-based method to mutate specified nucleotides of a sequence within a plasmid vector. This technique allows one to study the relative importance of a particular amino acid for protein structure and function. Typical mutations are designed to disrupt or map protein-protein interactions, mimic or block posttranslational modifications, or to silence enzymatic activity. Alternatively, noncoding changes are often used to generate rescue constructs that are resistant to knockdown via RNAi.

  17. 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.

  18. 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

  19. Chlordecone altered hepatic disposition of [{sup 14}C]cholesterol and plasma cholesterol distribution but not SR-BI or ABCG8 proteins in livers of C57BL/6 mice

    SciTech Connect

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

    2008-06-15

    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 [{sup 14}C]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 [{sup 14}C]CH by decreased hepatic and renal accumulation, and increased biliary excretion up to 300%. Biliary excretion of polar [{sup 14}C]CH metabolites was not altered by CD. CD pretreatment decreased subcellular distribution of [{sup 14}C]CH equivalents in hepatic cytosol and microsomes and lipoprotein-rich fraction-to-homogenate ratio. CD pretreatment increased the ratio of [{sup 14}C]CH equivalents in high density lipoprotein (HDL) to that in plasma and reduced [{sup 14}C]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 ATP-binding 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.

  20. 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

  1. 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

  2. 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

  3. 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.

  4. Empirical complexities in the genetic foundations of lethal mutagenesis.

    PubMed

    Bull, James J; Joyce, Paul; Gladstone, Eric; Molineux, Ian J

    2013-10-01

    From population genetics theory, elevating the mutation rate of a large population should progressively reduce average fitness. If the fitness decline is large enough, the population will go extinct in a process known as lethal mutagenesis. Lethal mutagenesis has been endorsed in the virology literature as a promising approach to viral treatment, and several in vitro studies have forced viral extinction with high doses of mutagenic drugs. Yet only one empirical study has tested the genetic models underlying lethal mutagenesis, and the theory failed on even a qualitative level. Here we provide a new level of analysis of lethal mutagenesis by developing and evaluating models specifically tailored to empirical systems that may be used to test the theory. We first quantify a bias in the estimation of a critical parameter and consider whether that bias underlies the previously observed lack of concordance between theory and experiment. We then consider a seemingly ideal protocol that avoids this bias-mutagenesis of virions-but find that it is hampered by other problems. Finally, results that reveal difficulties in the mere interpretation of mutations assayed from double-strand genomes are derived. Our analyses expose unanticipated complexities in testing the theory. Nevertheless, the previous failure of the theory to predict experimental outcomes appears to reside in evolutionary mechanisms neglected by the theory (e.g., beneficial mutations) rather than from a mismatch between the empirical setup and model assumptions. This interpretation raises the specter that naive attempts at lethal mutagenesis may augment adaptation rather than retard it.

  5. 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.

  6. 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

  7. Cancer gene discovery: exploiting insertional mutagenesis

    PubMed Central

    Ranzani, Marco; Annunziato, Stefano; Adams, David J.; Montini, Eugenio

    2013-01-01

    Insertional mutagenesis has been utilized as a functional forward genetics screen for the identification of novel genes involved in the pathogenesis of human cancers. Different insertional mutagens have been successfully used to reveal new cancer genes. For example, retroviruses (RVs) are integrating viruses with the capacity to induce the deregulation of genes in the neighborhood of the insertion site. RVs have been employed for more than 30 years to identify cancer genes in the hematopoietic system and mammary gland. Similarly, another tool that has revolutionized cancer gene discovery is the cut-and-paste transposons. These DNA elements have been engineered to contain strong promoters and stop cassettes that may function to perturb gene expression upon integration proximal to genes. In addition, complex mouse models characterized by tissue-restricted activity of transposons have been developed to identify oncogenes and tumor suppressor genes that control the development of a wide range of solid tumor types, extending beyond those tissues accessible using RV-based approaches. Most recently, lentiviral vectors (LVs) have appeared on the scene for use in cancer gene screens. LVs are replication defective integrating vectors that have the advantage of being able to infect non-dividing cells, in a wide range of cell types and tissues. In this review, we describe the various insertional mutagens focusing on their advantages/limitations and we discuss the new and promising tools that will improve the insertional mutagenesis screens of the future. PMID:23928056

  8. Site-directed mutagenesis of the CC chemokine binding protein 35K-Fc reveals residues essential for activity and mutations that increase the potency of CC chemokine blockade.

    PubMed

    White, Gemma E; McNeill, Eileen; Christou, Ivy; Channon, Keith M; Greaves, David R

    2011-08-01

    Chemokines of the CC class are key mediators of monocyte recruitment and macrophage differentiation and have a well documented role in many inflammatory diseases. Blockade of chemokine activity is therefore an attractive target for anti-inflammatory therapy. 35K (vCCI) is a high-affinity chemokine binding protein expressed by poxviruses, which binds all human and murine CC chemokines, preventing their interaction with chemokine receptors. We developed an Fc-fusion protein of 35K with a modified human IgG1 Fc domain and expressed this construct in human embryonic kidney 293T cells. Purified 35K-Fc is capable of inhibiting CC chemokine-induced calcium flux, chemotaxis, and β-arrestin recruitment in primary macrophages and transfected cells. To elucidate the residues involved in chemokine neutralization, we performed site-directed mutagenesis of six key amino acids in 35K and expressed the mutant Fc-fusion proteins in vitro. We screened the mutants for their ability to block chemokine-induced β-arrestin recruitment in transfected cells and to inhibit primary macrophage signaling in an electric cell substrate impedance sensing assay. Using a sterile model of acute inflammation, zymosan-induced peritonitis, we confirmed that wild-type 35K-Fc can reduce monocyte recruitment, whereas one mutant (R89A) showed a more pronounced blockade of monocyte influx and another mutant (E143K) showed total loss of function. We believe that 35K-Fc will be a useful tool for exploring the role of CC chemokines in chronic inflammatory pathologies, and we have identified a higher potency form of the molecule that may have potential therapeutic applications in chronic inflammatory disease.

  9. Lethal mutagenesis and evolutionary epidemiology.

    PubMed

    Martin, Guillaume; Gandon, Sylvain

    2010-06-27

    The lethal mutagenesis hypothesis states that within-host populations of pathogens can be driven to extinction when the load of deleterious mutations is artificially increased with a mutagen, and becomes too high for the population to be maintained. Although chemical mutagens have been shown to lead to important reductions in viral titres for a wide variety of RNA viruses, the theoretical underpinnings of this process are still not clearly established. A few recent models sought to describe lethal mutagenesis but they often relied on restrictive assumptions. We extend this earlier work in two novel directions. First, we derive the dynamics of the genetic load in a multivariate Gaussian fitness landscape akin to classical quantitative genetics models. This fitness landscape yields a continuous distribution of mutation effects on fitness, ranging from deleterious to beneficial (i.e. compensatory) mutations. We also include an additional class of lethal mutations. Second, we couple this evolutionary model with an epidemiological model accounting for the within-host dynamics of the pathogen. We derive the epidemiological and evolutionary equilibrium of the system. At this equilibrium, the density of the pathogen is expected to decrease linearly with the genomic mutation rate U. We also provide a simple expression for the critical mutation rate leading to extinction. Stochastic simulations show that these predictions are accurate for a broad range of parameter values. As they depend on a small set of measurable epidemiological and evolutionary parameters, we used available information on several viruses to make quantitative and testable predictions on critical mutation rates. In the light of this model, we discuss the feasibility of lethal mutagenesis as an efficient therapeutic strategy.

  10. Theory of lethal mutagenesis for viruses.

    PubMed

    Bull, J J; Sanjuán, R; Wilke, C O

    2007-03-01

    Mutation is the basis of adaptation. Yet, most mutations are detrimental, and elevating mutation rates will impair a population's fitness in the short term. The latter realization has led to the concept of lethal mutagenesis for curing viral infections, and work with drugs such as ribavirin has supported this perspective. As yet, there is no formal theory of lethal mutagenesis, although reference is commonly made to Eigen's error catastrophe theory. Here, we propose a theory of lethal mutagenesis. With an obvious parallel to the epidemiological threshold for eradication of a disease, a sufficient condition for lethal mutagenesis is that each viral genotype produces, on average, less than one progeny virus that goes on to infect a new cell. The extinction threshold involves an evolutionary component based on the mutation rate, but it also includes an ecological component, so the threshold cannot be calculated from the mutation rate alone. The genetic evolution of a large population undergoing mutagenesis is independent of whether the population is declining or stable, so there is no runaway accumulation of mutations or genetic signature for lethal mutagenesis that distinguishes it from a level of mutagenesis under which the population is maintained. To detect lethal mutagenesis, accurate measurements of the genome-wide mutation rate and the number of progeny per infected cell that go on to infect new cells are needed. We discuss three methods for estimating the former. Estimating the latter is more challenging, but broad limits to this estimate may be feasible.

  11. Arenavirus extinction through lethal mutagenesis.

    PubMed

    de la Torre, Juan Carlos

    2005-02-01

    Viral hemorrhagic fevers represent serious human public health problems causing devastating and often lethal disease. Several hemorrhagic fevers are caused by arenaviruses including Lassa fever virus (LFV) and the South American viral hemorrhagic fevers (SAHF). In recent years, increased air travel between Africa and other areas has led to the importation of LFV into the US, Europe, Japan, and Canada. This has raised awareness about arenaviruses as potential emerging viruses. Moreover, because of its severe morbidity and high mortality, and transmissibility from human to human, weaponized forms of LFV poses a real threat as agent of bioterrorism. No licensed vaccine is available in the US, and currently there is not efficacious therapy to treat these infections. Therefore, the importance of developing novel effective antiviral drugs to combat HF arenaviruses, for which the prototypic Arenavirus lymphocytic choriomeningitis virus (LCMV) provides us with an excellent model system. Recent findings have shown that LCMV multiplication both in cultured cells and in vivo is highly susceptible to the mutagenic agent 5-fluorouracil (FU). FU-mediated extinction of LCMV was associated with only modest increases in virus mutation frequencies, but did not significantly affect virus replication and transcription, or virus particle formation. These findings indicate that, as with other riboviruses, lethal mutagenesis is effective also against LCMV raising the possibility of using this novel antiviral strategy to combat pathogenic arenaviruses. PMID:15649566

  12. 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

  13. Highly efficient targeted mutagenesis in mice using TALENs.

    PubMed

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

    2013-11-01

    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.

  14. Spontaneous mutagenesis: experimental, genetic and other factors.

    PubMed

    Smith, K C

    1992-08-01

    Spontaneous mutations are "the net result of all that can go wrong with DNA during the life cycle of an organism" (Glickman et al., 1986). Thus, the types and amounts of spontaneous mutations produced are the resultant of all the cellular processes that are mutagenic and those that are antimutagenic. It is not widely appreciated that the types and frequencies of spontaneous mutations change markedly with subtle changes in experimental conditions. All types of mutations are produced spontaneously, i.e., base substitutions, frameshifts, insertions and deletions. However, very few papers have appeared that are devoted exclusively to the study of the mechanisms of spontaneous mutagenesis, and of the subtle experimental factors that affect the types and frequencies of spontaneous mutations. This is unfortunate because spontaneous mutagenesis appears to play a major role in evolution, aging, and carcinogenesis. This review emphasizes subtle experimental variables that markedly affect the results of a spontaneous mutation experiment. A thorough understanding of these variables eliminates the need for a theory of "directed" mutagenesis. The intrinsic instability of DNA, and the types of normal metabolic lesions that are produced in DNA that lead to mutations via errors made in replication, repair, and recombination are reviewed, as is the genetic control of spontaneous mutagenesis. As with spontaneous mutagenesis, spontaneous carcinogenesis can also be considered to be the net result of all that can go wrong with DNA during the life of an organism. PMID:1378531

  15. 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

  16. 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

  17. 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

  18. Therapeutically targeting RNA viruses via lethal mutagenesis.

    PubMed

    Graci, Jason D; Cameron, Craig E

    2008-11-01

    RNA viruses exhibit increased mutation frequencies relative to other organisms. Recent work has attempted to exploit this unique feature by increasing the viral mutation frequency beyond an extinction threshold, an antiviral strategy known as lethal mutagenesis. A number of novel nucleoside analogs have been designed around this premise. Herein, we review the quasispecies nature of RNA viruses and survey the antiviral, biological and biochemical characteristics of mutagenic nucleoside analogs, including clinically-used ribavirin. Biological implications of modulating viral replication fidelity are discussed in the context of translating lethal mutagenesis into a clinically-useful antiviral strategy.

  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. 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

  2. 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.

  3. DNA Polymerase ζ is essential for hexavalent chromium-induced mutagenesis

    PubMed Central

    O'Brien, Travis J.; Witcher, Preston; Brooks, Bradford; Patierno, Steven R.

    2009-01-01

    Translesion synthesis (TLS) is a unique DNA damage tolerance mechanism involved in the replicative bypass of genetic lesions in favor of uninterrupted DNA replication. TLS is critical for the generation of mutations by many different chemical and physical agents, however, there is no information available regarding the role of TLS in carcinogenic metal-induced mutagenesis. Hexavalent chromium (Cr(VI))-containing compounds are highly complex genotoxins possessing both mutagenic and clastogenic activities. The focus of this work was to determine the impact that TLS has on Cr(VI)-induced mutagenesis in S. cerevisiae. Wild-type yeast and strains deficient in TLS polymerases (i.e. Polζ (rev3), Polη (rad30)) were exposed to Cr(VI) and monitored for cell survival and forward mutagenesis at the CAN1 locus. In general, TLS deficiency had little impact on Cr(VI)-induced clonogenic lethality or cell growth. rad30 yeast exhibited higher levels of basal and induced mutagenesis compared to Wt and rev3 yeast. In contrast, rev3 yeast displayed attenuated Cr(VI)-induced mutagenesis. Moreover, deletion of REV3 in rad30 yeast (rad30 rev3) resulted in a significant decrease in basal and Cr(VI) mutagenesis relative to Wt and rad30 single mutants indicating that mutagenesis primarily depended upon Polζ. Interestingly, rev3 yeast were similar to Wt yeast in susceptibility to Cr(VI)-induced frameshift mutations. Mutational analysis of the CAN1 gene revealed that Cr(VI)-induced base substitution mutations accounted for 83.9% and 100.0% of the total mutations in Wt and rev3 yeast, respectively. Insertions and deletions comprised 16.1% of the total mutations in Cr(VI) treated Wt yeast but were not observed rev3 yeast. This work provides novel information regarding the molecular mechanisms of Cr(VI)-induced mutagenesis and is the first report demonstrating a role for TLS in the fixation of mutations induced by a carcinogenic metal. PMID:19428373

  4. 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.

  5. Fluorometric method of quantitative cell mutagenesis

    SciTech Connect

    Dolbeare, F.A.

    1982-08-17

    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.

  6. 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.

  7. 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

  8. [Evaluation of induced mutagenesis in workers engaged into chrysotile asbestos production].

    PubMed

    Zhumabekova, G S; Amanbekova, A U; Ibraeva, L K; Azhimetova, G N

    2014-01-01

    The authors present data of cytogenetic study of workers engaged into chrysotile asbestos industry. Evaluation of chromosomal aberrations in peripheral lymphocytes of workers in main workshops of "Kustanaiskie mineral" JSC revealed reliable increase in chromosomal aberrations level. Structural chromosomal abnormalities in main groups were presented by chromosome and chromatide type aberrations with latter prevelent--that can prove chemical mutagenesis. Chromosome type aberrations were presented by paired fragments and centromere rupture, those of chromatide type--by deletions, single fragments and chromatide ruptures. Higher values of induced mutagenesis were revealed in workers of chrysotile asbestos ore concentration workshop, in workers of ore-preparation workshop, and in individuals with over 25 years of work at chrysotile asbestos production.

  9. New mutations affecting induced mutagenesis in yeast.

    PubMed

    Lawrence, C W; Krauss, B R; Christensen, R B

    1985-01-01

    Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4-38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4+ and REV5+ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.

  10. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum.

    PubMed

    Gao, Junping; Wang, Genhong; Ma, Sanyuan; Xie, Xiaodong; Wu, Xiangwei; Zhang, Xingtan; Wu, Yuqian; Zhao, Ping; Xia, Qingyou

    2015-01-01

    Genome editing is one of the most powerful tools for revealing gene function and improving crop plants. Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been used as a powerful and efficient tool for genome editing in various organisms. Here, we report genome editing in tobacco (Nicotiana tabacum) mediated by the CRISPR/Cas9 system. Two genes, NtPDS and NtPDR6, were used for targeted mutagenesis. First, we examined the transient genome editing activity of this system in tobacco protoplasts, insertion and deletion (indel) mutations were observed with frequencies of 16.2-20.3% after transfecting guide RNA (gRNA) and the nuclease Cas9 in tobacco protoplasts. The two genes were also mutated using multiplexing gRNA at a time. Additionally, targeted deletions and inversions of a 1.8-kb fragment between two target sites in the NtPDS locus were demonstrated, while indel mutations were also detected at both the sites. Second, we obtained transgenic tobacco plants with NtPDS and NtPDR6 mutations induced by Cas9/gRNA. The mutation percentage was 81.8% for NtPDS gRNA4 and 87.5% for NtPDR6 gRNA2. Obvious phenotypes were observed, etiolated leaves for the psd mutant and more branches for the pdr6 mutant, indicating that highly efficient biallelic mutations occurred in both transgenic lines. No significant off-target mutations were obtained. Our results show that the CRISPR/Cas9 system is a useful tool for targeted mutagenesis of the tobacco genome.

  11. Predicting oligonucleotide-directed mutagenesis failures in protein engineering.

    PubMed

    Wassman, Christopher D; Tam, Phillip Y; Lathrop, Richard H; Weiss, Gregory A

    2004-01-01

    Protein engineering uses oligonucleotide-directed mutagenesis to modify DNA sequences through a two-step process of hybridization and enzymatic synthesis. Inefficient reactions confound attempts to introduce mutations, especially for the construction of vast combinatorial protein libraries. This paper applied computational approaches to the problem of inefficient mutagenesis. Several results implicated oligonucleotide annealing to non-target sites, termed 'cross-hybridization', as a significant contributor to mutagenesis reaction failures. Test oligonucleotides demonstrated control over reaction outcomes. A novel cross-hybridization score, quickly computable for any plasmid and oligonucleotide mixture, directly correlated with yields of deleterious mutagenesis side products. Cross-hybridization was confirmed conclusively by partial incorporation of an oligonucleotide at a predicted cross-hybridization site, and by modification of putative template secondary structure to control cross-hybridization. Even in low concentrations, cross-hybridizing species in mixtures poisoned reactions. These results provide a basis for improved mutagenesis efficiencies and increased diversities of cognate protein libraries.

  12. Stress-induced mutagenesis and complex adaptation

    PubMed Central

    Ram, Yoav; Hadany, Lilach

    2014-01-01

    Because mutations are mostly deleterious, mutation rates should be reduced by natural selection. However, mutations also provide the raw material for adaptation. Therefore, evolutionary theory suggests that the mutation rate must balance between adaptability—the ability to adapt—and adaptedness—the ability to remain adapted. We model an asexual population crossing a fitness valley and analyse the rate of complex adaptation with and without stress-induced mutagenesis (SIM)—the increase of mutation rates in response to stress or maladaptation. We show that SIM increases the rate of complex adaptation without reducing the population mean fitness, thus breaking the evolutionary trade-off between adaptability and adaptedness. Our theoretical results support the hypothesis that SIM promotes adaptation and provide quantitative predictions of the rate of complex adaptation with different mutational strategies. PMID:25143032

  13. 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.

  14. 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.

  15. 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

  16. Scoring function to predict solubility mutagenesis

    PubMed Central

    2010-01-01

    Background Mutagenesis is commonly used to engineer proteins with desirable properties not present in the wild type (WT) protein, such as increased or decreased stability, reactivity, or solubility. Experimentalists often have to choose a small subset of mutations from a large number of candidates to obtain the desired change, and computational techniques are invaluable to make the choices. While several such methods have been proposed to predict stability and reactivity mutagenesis, solubility has not received much attention. Results We use concepts from computational geometry to define a three body scoring function that predicts the change in protein solubility due to mutations. The scoring function captures both sequence and structure information. By exploring the literature, we have assembled a substantial database of 137 single- and multiple-point solubility mutations. Our database is the largest such collection with structural information known so far. We optimize the scoring function using linear programming (LP) methods to derive its weights based on training. Starting with default values of 1, we find weights in the range [0,2] so that predictions of increase or decrease in solubility are optimized. We compare the LP method to the standard machine learning techniques of support vector machines (SVM) and the Lasso. Using statistics for leave-one-out (LOO), 10-fold, and 3-fold cross validations (CV) for training and prediction, we demonstrate that the LP method performs the best overall. For the LOOCV, the LP method has an overall accuracy of 81%. Availability Executables of programs, tables of weights, and datasets of mutants are available from the following web page: http://www.wsu.edu/~kbala/OptSolMut.html. PMID:20929563

  17. 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

    2016-07-11

    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.

  18. 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

  19. Insertion and deletion mutagenesis of the human cytomegalovirus genome

    SciTech Connect

    Spaete, R.R.; Mocarski, E.S.

    1987-10-01

    Studies on human cytomegalovirus (CMV) have been limited by a paucity of molecular genetic techniques available for manipulating the viral genome. The authors have developed methods for site-specific insertion and deletion mutagenesis of CMV utilizing a modified Escherichia coli lacZ gene as a genetic marker. The lacZ gene was placed under the control of the major ..beta.. gene regulatory signals and inserted into the viral genome by homologous recombination, disrupting one of two copies of this ..beta.. gene within the L-component repeats of CMV DNA. They observed high-level expression of ..beta..-galactosidase by the recombinant in a temporally authentic manner, with levels of this enzyme approaching 1% of total protein in infected cells. Thus, CMV is an efficient vector for high-level expression of foreign gene products in human cells. Using back selection of lacZ-deficient virus in the presence of the chromogenic substrate 5-bromo-4-chloro-3-indolyl ..beta..-D-galactoside, they generated random endpoint deletion mutants. Analysis of these mutant revealed that CMV DNA sequences flanking the insert had been removed, thereby establishing this approach as a means of determining whether sequences flanking a lacZ insertion are dispensable for viral growth. In an initial test of the methods, they have shown that 7800 base pairs of one copy of L-component repeat sequences can be deleted without affecting viral growth in human fibroblasts.

  20. History of attempts to quantify environmental mutagenesis

    SciTech Connect

    Hollaender, A.

    1981-01-01

    It became obvious in the early 1960's that the ready recognition of mutations produced by chemicals could have a profound influence on the refinement of methods to detect environmental mutagens. The experience derived over the previous 30 years in characterizing the effects of ionizing and ultraviolet radiation on the genetic mechanism came to serve us in good stead. Although the effects of chemicals are considerably more complicated and often require the analysis of individual substances, nonetheless, the area has developed rapidly in recent decades. The establishment and historical background of the International Association of Environmental Mutagen Societies (IAEMS) will be discussed. An attempt at the quantitation of chemical effects has been developed in comparison with radiation mutagenesis. As a first step, a definition of the Mutagen Burden or unavoidable exposure to chemicals will be discussed. A mathematical approach (Haynes/Eckhardt) will be considered and finally an outline for the comprehensive investigation of detailed interscience study will be made of less than six chemicals.

  1. 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

  2. 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).

  3. Targeted mutagenesis tools for modelling psychiatric disorders.

    PubMed

    Deussing, Jan M

    2013-10-01

    In the 1980s, the basic principles of gene targeting were discovered and forged into sharp tools for efficient and precise engineering of the mouse genome. Since then, genetic mouse models have substantially contributed to our understanding of major neurobiological concepts and are of utmost importance for our comprehension of neuropsychiatric disorders. The "domestication" of site-specific recombinases and the continuous creative technological developments involving the implementation of previously identified biological principles such as transcriptional and posttranslational control now enable conditional mutagenesis with high spatial and temporal resolution. The initiation and successful accomplishment of large-scale efforts to annotate functionally the entire mouse genome and to build strategic resources for the research community have significantly accelerated the rapid proliferation and broad propagation of mouse genetic tools. Addressing neurobiological processes with the assistance of genetic mouse models is a routine procedure in psychiatric research and will be further extended in order to improve our understanding of disease mechanisms. In light of the highly complex nature of psychiatric disorders and the current lack of strong causal genetic variants, a major future challenge is to model of psychiatric disorders more appropriately. Humanized mice, and the recently developed toolbox of site-specific nucleases for more efficient and simplified tailoring of the genome, offer the perspective of significantly improved models. Ultimately, these tools will push the limits of gene targeting beyond the mouse to allow genome engineering in any model organism of interest.

  4. Favipiravir elicits antiviral mutagenesis during virus replication in vivo.

    PubMed

    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.

  5. 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

  6. 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)

  7. 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

  8. 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

  9. 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

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

    PubMed

    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.

  11. Segment-specific mutagenesis: extensive mutagenesis of a lac promoter/operator element.

    PubMed

    Weiher, H; Schaller, H

    1982-03-01

    A method for highly efficient segment-specific mutagenesis is described. The method uses as target for sodium bisulfite mutagenesis the DNA single strands of a DNA restriction fragment that had been separated by cloning into base-complementary regions of a pair of phage fd vectors. After repair synthesis in vitro, the mutagenized DNA fragment is recovered by cloning into a nonmutated plasmid vector and analyzed for sequence and by functional tests. By using this method, the nucleotide sequence of a 109-base pair restriction fragment containing the lac promoter/operator from Escherichia coli was extensively modified. More than 90% of the 235 isolates obtained showed a change in phenotype; all of 22 analyzed for their nucleotide sequence were found to carry multiple C leads to T point mutations in up to 60% of the possible target positions. Nevertheless, few isolates showed major changes in promoter activity relative to the nonmutated promoter element, which indicates a high degree of flexibility in the promoter sequence. PMID:7041119

  12. [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

  13. 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.

  14. Stabilization of a prokaryotic LAT transporter by random mutagenesis.

    PubMed

    Rodríguez-Banqueri, Arturo; Errasti-Murugarren, Ekaitz; Bartoccioni, Paola; Kowalczyk, Lukasz; Perálvarez-Marín, Alex; Palacín, Manuel; Vázquez-Ibar, José Luis

    2016-04-01

    The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis L-serine/L-threonine exchanger is the best-known prokaryotic paradigm of the mammalian L-amino acid transporter (LAT) family. Unfortunately, SteT's lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest. PMID:26976827

  15. Catalytic efficiency of expressed aromatase following site-directed mutagenesis.

    PubMed

    Kadohama, N; Zhou, D; Chen, S; Osawa, Y

    1993-05-13

    Mutant aromatase cytochrome P-450s, expressed in CHO cells after transfection with cDNAs, have been characterized in terms of their catalytic efficiencies. After solubilization from microsomes, specific aromatase P-450 content of wild-type and mutants Pro308Phe, Asp309Asn, Asp309Ala and Phe406Arg was quantitated by a sandwich enzyme-linked immunosorbent assay (ELISA). Microsomal aromatase activity was determined by the 3H-water method using [1 beta-3H]androstenedione as substrate. Estimations of the actual turnover rate (catalytic efficiency) were derived from the combined data. The P-450 content in the mutants varied but was always less than that in the wild type. Hence, the decreases in the Vmax observed in the mutant enzymes did not correlate completely with reductions in catalytic effectiveness. In recent studies on the structure-function relationship of aromatase cytochrome P-450, the observed reduction of enzyme activity in terms of Vmax following site-directed mutagenesis led to the assumption that there was a corresponding loss of catalytic effectiveness. The present study reveals that a lower P-450 content can contribute significantly to decreasing catalytic activity in the mutants. In fact, in mutant Phe406Arg which exhibited virtually no catalytically active aromatase, the specific P-450 content was below the detectable level. Because of its location, the result of this latter mutation could be a major structural perturbation of the heme-binding property. Thus, interpretation of losses and reductions in aromatase activity resulting from single amino-acid replacement should take into account changes in the specific content of aromatase cytochrome P-450.

  16. CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

    PubMed

    Xue, Zhaoyu; Wu, Menghua; Wen, Kejia; Ren, Menda; Long, Li; Zhang, Xuedi; Gao, Guanjun

    2014-11-01

    Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila. Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach. PMID:25193494

  17. Random tag insertions by Transposon Integration mediated Mutagenesis (TIM).

    PubMed

    Hoeller, Brigitte M; Reiter, Birgit; Abad, Sandra; Graze, Ina; Glieder, Anton

    2008-10-01

    Transposon Integration mediated Mutagenesis (TIM) is a broadly applicable tool for protein engineering. This method combines random integration of modified bacteriophage Mu transposons with their subsequent defined excision employing type IIS restriction endonuclease AarI. TIM enables deletion or insertion of an arbitrary number of bases at random positions, insertion of functional sequence tags at random positions, replacing randomly selected triplets by a specific codon (e.g. scanning) and site-saturation mutagenesis. As a proof of concept a transposon named GeneOpenerAarIKan was designed and employed to introduce 6xHis tags randomly into the esterase EstC from Burkholderia gladioli. A TIM library was screened with colony based assays for clones with an integrated 6xHis tag and for clones exhibiting esterase activity. The employed strategy enables the isolation of randomly tagged active enzymes in single mutagenesis experiments.

  18. 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

  19. Theories of Lethal Mutagenesis: From Error Catastrophe to Lethal Defection.

    PubMed

    Tejero, Héctor; Montero, Francisco; Nuño, Juan Carlos

    2016-01-01

    RNA viruses get extinct in a process called lethal mutagenesis when subjected to an increase in their mutation rate, for instance, by the action of mutagenic drugs. Several approaches have been proposed to understand this phenomenon. The extinction of RNA viruses by increased mutational pressure was inspired by the concept of the error threshold. The now classic quasispecies model predicts the existence of a limit to the mutation rate beyond which the genetic information of the wild type could not be efficiently transmitted to the next generation. This limit was called the error threshold, and for mutation rates larger than this threshold, the quasispecies was said to enter into error catastrophe. This transition has been assumed to foster the extinction of the whole population. Alternative explanations of lethal mutagenesis have been proposed recently. In the first place, a distinction is made between the error threshold and the extinction threshold, the mutation rate beyond which a population gets extinct. Extinction is explained from the effect the mutation rate has, throughout the mutational load, on the reproductive ability of the whole population. Secondly, lethal defection takes also into account the effect of interactions within mutant spectra, which have been shown to be determinant for the understanding the extinction of RNA virus due to an augmented mutational pressure. Nonetheless, some relevant issues concerning lethal mutagenesis are not completely understood yet, as so survival of the flattest, i.e. the development of resistance to lethal mutagenesis by evolving towards mutationally more robust regions of sequence space, or sublethal mutagenesis, i.e., the increase of the mutation rate below the extinction threshold which may boost the adaptability of RNA virus, increasing their ability to develop resistance to drugs (including mutagens). A better design of antiviral therapies will still require an improvement of our knowledge about lethal

  20. ENU mutagenesis to generate genetically modified rat models.

    PubMed

    van Boxtel, Ruben; Gould, Michael N; Cuppen, Edwin; Smits, Bart M G

    2010-01-01

    The rat is one of the most preferred model organisms in biomedical research and has been extremely useful for linking physiology and pathology to the genome. However, approaches to genetically modify specific genes in the rat germ line remain relatively scarce. To date, the most efficient approach for generating genetically modified rats has been the target-selected N-ethyl-N-nitrosourea (ENU) mutagenesis-based technology. Here, we describe the detailed protocols for ENU mutagenesis and mutant retrieval in the rat model organism.

  1. 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

  2. Mutagenesis of Trichoderma Viride by Ultraviolet and Plasma

    NASA Astrophysics Data System (ADS)

    Yao, Risheng; Li, Manman; Deng, Shengsong; Hu, Huajia; Wang, Huai; Li, Fenghe

    2012-04-01

    Considering the importance of a microbial strain capable of increased cellulase production, a mutant strain UP4 of Trichoderma viride was developed by ultraviolet (UV) and plasma mutation. The mutant produced a 21.0 IU/mL FPase which was 98.1% higher than that of the parent strain Trichoderma viride ZY-1. In addition, the effect of ultraviolet and plasma mutagenesis was not merely simple superimposition of single ultraviolet mutation and single plasma mutation. Meanwhile, there appeared a capsule around some of the spores after the ultraviolet and plasma treatment, namely, the spore surface of the strain became fuzzy after ultraviolet or ultraviolet and plasma mutagenesis.

  3. 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

  4. Meiotic chromosome segregation mutants identified by insertional mutagenesis of fission yeast Schizosaccharomyces pombe; tandem-repeat, single-site integrations

    PubMed Central

    Davidson, Mari K.; Young, Nathan P.; Glick, Gloria G.; Wahls, Wayne P.

    2004-01-01

    Identification of genes required for segregation of chromosomes in meiosis (scm) is difficult because in most organisms high-fidelity chromosome segregation is essential to produce viable meiotic products. The biology of fission yeast Schizosaccharomyces pombe facilitates identification of such genes. Insertional mutagenesis was achieved by electroporation of linear ura4+ DNA into cells harboring a ura4 deletion. Approximately 1000 stable transformants were screened individually for the production of elevated frequencies of aneuploid spore colonies. Twenty-two candidates were subjected to a secondary screen for cytological defects. Five mutants exhibited significant levels of aberrant meiotic chromosome segregation, but were proficient for mating and completion of meiosis. Each mutant's phenotype cosegregated with its respective ura4+ transgene. The mutations were recessive and defined five complementation groups, revealing five distinct genes (scm1, scm2, scm3, scm4 and scm5). Southern blotting revealed single-site integration in each transformant, indicating that insertional mutagenesis is useful for generating single-locus scm mutations linked to a selectable marker. The transgene insertion points were refractory to analysis by inverse-PCR. Molecular and real-time PCR analyses revealed the presence of multiple, truncated copies of ura4+ at each integration site. Thus, electroporation-mediated insertional mutagenesis in S.pombe is preceded by exonucleolytic processing and concatomerization of the transforming DNA. PMID:15316103

  5. Meiotic chromosome segregation mutants identified by insertional mutagenesis of fission yeast Schizosaccharomyces pombe; tandem-repeat, single-site integrations.

    PubMed

    Davidson, Mari K; Young, Nathan P; Glick, Gloria G; Wahls, Wayne P

    2004-01-01

    Identification of genes required for segregation of chromosomes in meiosis (scm) is difficult because in most organisms high-fidelity chromosome segregation is essential to produce viable meiotic products. The biology of fission yeast Schizosaccharomyces pombe facilitates identification of such genes. Insertional mutagenesis was achieved by electroporation of linear ura4+ DNA into cells harboring a ura4 deletion. Approximately 1000 stable transformants were screened individually for the production of elevated frequencies of aneuploid spore colonies. Twenty-two candidates were subjected to a secondary screen for cytological defects. Five mutants exhibited significant levels of aberrant meiotic chromosome segregation, but were proficient for mating and completion of meiosis. Each mutant's phenotype cosegregated with its respective ura4+ transgene. The mutations were recessive and defined five complementation groups, revealing five distinct genes (scm1, scm2, scm3, scm4 and scm5). Southern blotting revealed single-site integration in each transformant, indicating that insertional mutagenesis is useful for generating single-locus scm mutations linked to a selectable marker. The transgene insertion points were refractory to analysis by inverse-PCR. Molecular and real-time PCR analyses revealed the presence of multiple, truncated copies of ura4+ at each integration site. Thus, electroporation-mediated insertional mutagenesis in S.pombe is preceded by exonucleolytic processing and concatomerization of the transforming DNA. PMID:15316103

  6. 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

  7. 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

  8. 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.

  9. Implementing large-scale ENU mutagenesis screens in North America

    PubMed Central

    Clark, Amander T.; Goldowitz, Daniel; Takahashi, Joseph S.; Vitaterna, Martha Hotz; Siepka, Sandra M.; Peters, Luanne L.; Frankel, Wayne N.; Carlson, George A.; Rossant, Janet; Nadeau, Joseph H.; Justice, Monica J.

    2013-01-01

    A step towards annotating the mouse genome is to use forward genetics in phenotype-driven screens to saturate the genome with mutations. The purpose of this article is to highlight the new projects in North America that are focused on isolating mouse mutations after ENU mutagenesis and phenotype screening. PMID:15619961

  10. 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

  11. 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

  12. 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...

  13. 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…

  14. Suitability of tamoxifen-induced mutagenesis for behavioral phenotyping.

    PubMed

    Vogt, M A; Chourbaji, S; Brandwein, C; Dormann, C; Sprengel, R; Gass, P

    2008-05-01

    Tamoxifen-induced mutagenesis via the so-called CreER(T2) fusion enzyme is a key technology for the inducible gene knockout in the adult murine brain. However, it requires a subchronic transient treatment with high doses of the non-selective estrogen receptor antagonist tamoxifen. It has been shown earlier that acute tamoxifen treatment causes behavioral alterations, while the long-term behavioral effects of tamoxifen in mice are so far unknown. Therefore C57BL/6 male mice, a common strain used for targeted mutagenesis and behavioral analyses, were subjected to a tamoxifen treatment protocol as used for inducible mutagenesis in vivo, and analyzed for effects on general behavior (locomotion, exploration), emotional behavior (anxiety, depression) and on learning and memory after a drug-free interval period of 4 weeks. The results demonstrate that a test for depression-like behavior, i.e. the Forced Swim Test, is affected even more than 4 weeks after tamoxifen treatment. In contrast, in all other tests, tamoxifen treated mice showed unaltered behaviors, indicating that the currently established 5-day protocol of tamoxifen treatment (40 mg/kg bid) for inducible mutagenesis has no or little effects on the behavior of C57BL/6 male mice after a latency period of 4 weeks. These results are important for all studies using tamoxifen-induced mutagenesis since this protocol obviously does not evoke alterations in general behaviors such as locomotion, exploration or anxiety-like behaviors, which might confound more complex behavioral analyses, nor does it affect standard tests for learning and memory, such as Morris Water Maze, contextual and cued Fear Conditioning and T-Maze learning.

  15. 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.

  16. 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

  17. Natural mutagenesis of human genomes by endogenous retrotransposons

    PubMed Central

    Iskow, Rebecca C.; McCabe, Michael T.; Mills, Ryan E.; Torene, Spencer; Pittard, W. Stephen; Neuwald, Andrew F.; Van Meir, Erwin G.; Vertino, Paula M.; Devine, Scott E.

    2010-01-01

    SUMMARY Two abundant classes of mobile elements, namely Alu and L1 elements, continue to generate new retrotransposon insertions in human genomes. Estimates suggest that these elements have generated millions of new germline insertions in individual human genomes worldwide. Unfortunately, current technologies are not capable of detecting most of these young insertions, and the true extent of germline mutagenesis by endogenous human retrotransposons has been difficult to examine. Here, we describe new technologies for detecting these young retrotransposon insertions and demonstrate that such insertions indeed are abundant in human populations. We also found that new somatic L1 insertions occur at high frequencies in human lung cancer genomes. Genome-wide analysis suggests that altered DNA methylation may be responsible for the high levels of L1 mobilization observed in these tumors. Our data indicate that transposon-mediated mutagenesis is extensive in human genomes, and is likely to have a major impact on human biology and diseases. PMID:20603005

  18. Nonrandom mutagenesis. Progress report, March 1, 1981-February 28, 1982

    SciTech Connect

    Goldsby, R.A.

    1981-01-01

    The ultimate goal is the development of tools, approaches and systems which will increase our ability to detect and control mutagenesis. We have continued to develop hybrid cell lines suited to the investigation of the expression and mutagenesis of human cell surface markers. The development and characterization of the monoclonal antibody probes to identify and characterize variation in selected human cell surface antigens has continued. Human X mouse T lymphoma hybrids have proven valuable in obtaining clonal populations expressing cell surface determinants characteristic of particular differentiated cell types. We have constructed a set of human lymphocyte X mouse T lymphoma hybrids which have proven useful for the mapping of cell surface determinants to particular chromosomes.

  19. 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

  20. 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.

  1. Topology of transmembrane proteins by scanning cysteine accessibility mutagenesis methodology.

    PubMed

    Zhu, Quansheng; Casey, Joseph R

    2007-04-01

    Integral membrane proteins of the plasma membrane span from the inside to the outside of the cell. The primary structural element of integral membrane proteins is their topology: the pattern in which the protein traverses the membrane. A full description of topology, defining which parts of the protein face outside versus inside, goes a long way toward understanding the folding of these proteins. Many approaches have been established to define membrane protein topology. Here, we present the technique of scanning cysteine accessibility mutagenesis (SCAM). This approach uses the unique chemical reactivity of the cysteine sulfhydryl to probe membrane protein structure. Individual cysteine residues are introduced into the target protein by mutagenesis. The ability to chemically react these residues using sulfhydryl-directed reagents (either membrane permeant or impermeant) defines each site as either extracellular or intracellular, thus establishing topology of a location. This analysis performed on many sites in the protein will define the protein's topology. PMID:17367716

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue▿

    PubMed Central

    Graci, Jason D.; Harki, Daniel A.; Korneeva, Victoria S.; Edathil, Jocelyn P.; Too, Kathleen; Franco, David; Smidansky, Eric D.; Paul, Aniko V.; Peterson, Blake R.; Brown, Daniel M.; Loakes, David; Cameron, Craig E.

    2007-01-01

    Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase-mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension, and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous base-pairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture, owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses. PMID:17686844

  8. Mutagenesis by the autoxidation of iron with isolated DNA

    SciTech Connect

    Loeb, L.A.; James, E.A.; Waltersdorph, A.M.; Klebanoff, S.J. )

    1988-06-01

    Oxygen free radicals are highly reactive species generated by many cellular oxidation-reduction processes. These radicals damage cellular constituents and have been casually implicated in the pathogenesis of many human diseases. The authors report here that oxygen free radicals generated by Fe{sup 2+} in aqueous solution are mutagenic. Aerobic incubation of {phi}X174 am3 (amber 3 mutation) DNA with Fe{sup 2+} results in decreased phage survival when the treated DNA is transfected into Escherichia coli spheroplasts. Transfection of the treated DNA into SOS-induced spheroplasts results in an increase in mutagenesis as great as 50-fold. Both killing and mutagenesis can be prevented by binding of Fe{sup 2+} with deferoxamine or by the addition of catalase or mannitol. These results suggest that DNA damage and mutagenesis brought about by Fe{sup 2+} are likely to occur by a Fenton-type mechanism. DNA sequence analysis of the Fe{sup 2+}-induced mutants indicates that reversion of the phage phenotype to wild type occurs largely by a transversion type of mutation involving substitution of deoxyadenosine for thymidine opposite a template deoxyadenosine. These findings raise the possibility that free iron localized in cellular DNA may cause mutations by the generation of oxygen free radicals.

  9. Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

    PubMed

    Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

    2016-01-01

    A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

  10. The role of flexibility and molecular shape in the crystallization of proteins by surface mutagenesis.

    PubMed

    Devedjiev, Yancho D

    2015-02-01

    Proteins are dynamic systems and interact with their environment. The analysis of crystal contacts in the most accurately determined protein structures (d < 1.5 Å) reveals that in contrast to current views, static disorder and high side-chain entropy are common in the crystal contact area. These observations challenge the validity of the theory that presumes that the occurrence of well ordered patches of side chains at the surface is an essential prerequisite for a successful crystallization event. The present paper provides evidence in support of the approach for understanding protein crystallization as a process dependent on multiple factors, each with its relative contribution, rather than a phenomenon driven by a few dominant physicochemical characteristics. The role of the molecular shape as a factor in the crystallization of proteins by surface mutagenesis is discussed.

  11. 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.

  12. 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.

  13. 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

  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 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

  16. 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.

  17. [Influence of diethyl sulfate (DES) mutagenesis on growth properties and pigment secondary metabolites of Phellinus igniarius].

    PubMed

    Wang, Jing; Wu, Xin-yuan; Ma, Wei; Chen, Jing; Liu, Cheng; Wu, Xiu-li

    2015-06-01

    The diethyl sulfate (DES) mutagenesis was chosen for the mutagenic treatment to Phellinus igniarius, and the relationship of mutagenesis time and death rate was investigated with 0.5% DES. The differences of mycelial growth speed, liquid fermentation mycelia biomass, morphology and pigment classes of secondary metabolites production speed and antioxidant activities of metabolite products were discussed. The study displayed that DES mutagenesis could change mycelial morphology without obvious effect on mycelium growth, and the DES mutagenesis improved antioxidant activities of the active ingredients of P. igniarius and had more antioxidant activity of hypoxia/sugar PC12 nerve cells than that of P. igniarius. PMID:26591512

  18. 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

  19. 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.

  20. 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

  1. Functional Consequence of Positive Selection Revealed through Rational Mutagenesis of Human Myeloperoxidase

    PubMed Central

    Loughran, Noeleen B.; Hinde, Sara; McCormick-Hill, Sally; Leidal, Kevin G.; Bloomberg, Sarah; Loughran, Sinéad T.; O’Connor, Brendan; Ó'Fágáin, Ciarán; Nauseef, William M.; O’Connell, Mary J.

    2012-01-01

    Myeloperoxidase (MPO) is a member of the mammalian heme peroxidase (MHP) multigene family. Whereas all MHPs oxidize specific halides to generate the corresponding hypohalous acid, MPO is unique in its capacity to oxidize chloride at physiologic pH to produce hypochlorous acid (HOCl), a potent microbicide that contributes to neutrophil-mediated host defense against infection. We have previously resolved the evolutionary relationships in this functionally diverse multigene family and predicted in silico that positive Darwinian selection played a major role in the observed functional diversities (Loughran NB, O'Connor B, O'Fagain C, O'Connell MJ. 2008. The phylogeny of the mammalian heme peroxidases and the evolution of their diverse functions. BMC Evol Biol. 8:101). In this work, we have replaced positively selected residues asparagine 496 (N496), tyrosine 500 (Y500), and leucine 504 (L504) with the amino acids present in the ancestral MHP and have examined the effects on the structure, biosynthesis, and activity of MPO. Analysis in silico predicted that N496F, Y500F, or L504T would perturb hydrogen bonding in the heme pocket of MPO and thus disrupt the structural integrity of the enzyme. Biosynthesis of the mutants stably expressed in human embryonic kidney 293 cells yielded apoproMPO, the heme-free, enzymatically inactive precursor of MPO, that failed to undergo normal maturation or proteolytic processing. As a consequence of the maturational arrest at the apoproMPO stage of development, cells expressing MPO with mutations N496F, Y500F, L504T, individually or in combination, lacked normal peroxidase or chlorinating activity. Taken together, our data provide further support for the in silico predictions of positive selection and highlight the correlation between positive selection and functional divergence. Our data demonstrate that directly probing the functional importance of positive selection can provide important insights into understanding protein evolution. PMID:22355012

  2. Proline Scanning Mutagenesis Reveals a Role for the Flap Endonuclease-1 Helical Cap in Substrate Unpairing*

    PubMed Central

    Patel, Nikesh; Exell, Jack C.; Jardine, Emma; Ombler, Ben; Finger, L. David; Ciani, Barbara; Grasby, Jane A.

    2013-01-01

    The prototypical 5′-nuclease, flap endonuclease-1 (FEN1), catalyzes the essential removal of single-stranded flaps during DNA replication and repair. FEN1 hydrolyzes a specific phosphodiester bond one nucleotide into double-stranded DNA. This specificity arises from double nucleotide unpairing that places the scissile phosphate diester on active site divalent metal ions. Also related to FEN1 specificity is the helical arch, through which 5′-flaps, but not continuous DNAs, can thread. The arch contains basic residues (Lys-93 and Arg-100 in human FEN1 (hFEN1)) that are conserved by all 5′-nucleases and a cap region only present in enzymes that process DNAs with 5′ termini. Proline mutations (L97P, L111P, L130P) were introduced into the hFEN1 helical arch. Each mutation was severely detrimental to reaction. However, all proteins were at least as stable as wild-type (WT) hFEN1 and bound substrate with comparable affinity. Moreover, all mutants produced complexes with 5′-biotinylated substrate that, when captured with streptavidin, were resistant to challenge with competitor DNA. Removal of both conserved basic residues (K93A/R100A) was no more detrimental to reaction than the single mutation R100A, but much less severe than L97P. The ability of protein-Ca2+ to rearrange 2-aminopurine-containing substrates was monitored by low energy CD. Although L97P and K93A/R100A retained the ability to unpair substrates, the cap mutants L111P and L130P did not. Taken together, these data challenge current assumptions related to 5′-nuclease family mechanism. Conserved basic amino acids are not required for double nucleotide unpairing and appear to act cooperatively, whereas the helical cap plays an unexpected role in hFEN1-substrate rearrangement. PMID:24126913

  3. 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.

  4. Lethal Mutagenesis of Hepatitis C Virus Induced by Favipiravir

    PubMed Central

    de Ávila, Ana I.; Gallego, Isabel; Soria, Maria Eugenia; Gregori, Josep; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M.; Domingo, Esteban; Perales, Celia

    2016-01-01

    Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagen. Here we show that favipiravir (T-705) is a potent mutagenic agent for hepatitis C virus (HCV) during its replication in human hepatoma cells. T-705 leads to an excess of G → A and C → U transitions in the mutant spectrum of preextinction HCV populations. Infectivity decreased significantly in the presence of concentrations of T-705 which are 2- to 8-fold lower than its cytotoxic concentration 50 (CC50). Passaging the virus five times in the presence of 400 μM T-705 resulted in virus extinction. Since T-705 has undergone advanced clinical trials for approval for human use, the results open a new approach based on lethal mutagenesis to treat hepatitis C virus infections. If proven effective for HCV in vivo, this new anti-HCV agent may be useful in patient groups that fail current therapeutic regimens. PMID:27755573

  5. A mariner transposon vector adapted for mutagenesis in oral streptococci

    PubMed Central

    Nilsson, Martin; Christiansen, Natalia; Høiby, Niels; Twetman, Svante; Givskov, Michael; Tolker-Nielsen, Tim

    2014-01-01

    This article describes the construction and characterization of a mariner-based transposon vector designed for use in oral streptococci, but with a potential use in other Gram-positive bacteria. The new transposon vector, termed pMN100, contains the temperature-sensitive origin of replication repATs-pWV01, a selectable kanamycin resistance gene, a Himar1 transposase gene regulated by a xylose-inducible promoter, and an erythromycin resistance gene flanked by himar inverted repeats. The pMN100 plasmid was transformed into Streptococcus mutans UA159 and transposon mutagenesis was performed via a protocol established to perform high numbers of separate transpositions despite a low frequency of transposition. The distribution of transposon inserts in 30 randomly picked mutants suggested that mariner transposon mutagenesis is unbiased in S. mutans. A generated transposon mutant library containing 5000 mutants was used in a screen to identify genes involved in the production of sucrose-dependent extracellular matrix components. Mutants with transposon inserts in genes encoding glycosyltransferases and the competence-related secretory locus were predominantly found in this screen. PMID:24753509

  6. 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

  7. TALEN mediated somatic mutagenesis in murine models of cancer

    PubMed Central

    Zhang, Shuyuan; Li, Lin; Kendrick, Sara L.; Gerard, Robert D.; Zhu, Hao

    2014-01-01

    Cancer genome sequencing has identified numerous somatic mutations whose biological relevance is uncertain. In this study, we used genome-editing tools to create and analyze targeted somatic mutations in murine models of liver cancer. TALEN were designed against β-catenin (Ctnnb1) and Apc, two commonly mutated genes in hepatocellular carcinoma (HCC), to generate isogenic HCC cell lines. Both mutant cell lines exhibited evidence of Wnt pathway dysregulation. We asked if these TALENs could create targeted somatic mutations after hydrodynamic transfection (HDT) into mouse liver. TALENs targeting β-catenin promoted endogenous HCC carrying the intended gain-of-function mutations. However, TALENs targeting Apc were not as efficient in inducing in vivo homozygous loss-of-function mutations. We hypothesized that hepatocyte polyploidy might be protective against TALEN-induced loss of heterozygosity (LOH), and indeed Apc gene editing was less efficient in tetraploid than in diploid hepatocytes. To increase efficiency, we administered adenoviral Apc TALENs and found that we could achieve a higher mutagenesis rate in vivo. Our results demonstrate that genome-editing tools can enable the in vivo study of cancer genes and faithfully recapitulate the mosaic nature of mutagenesis in mouse cancer models. PMID:25070752

  8. The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation).

    PubMed

    Maisnier-Patin, Sophie; Roth, John R

    2015-07-01

    Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac(+)) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F'lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F'lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac(+) triggers exponential cell growth leading to a stable Lac(+) revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac(+) colony. Cells with multiple copies of the F'lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac(+) revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns-Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions.

  9. 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

  10. 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...

  11. 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.

  12. 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

  13. 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

  14. 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)

  15. Error-prone rolling circle amplification greatly simplifies random mutagenesis.

    PubMed

    Fujii, Ryota; Kitaoka, Motomitsu; Hayashi, Kiyoshi

    2014-01-01

    We describe a simple and easy protocol to introduce random mutations into plasmid DNA: error-prone rolling circle amplification. A template plasmid is amplified via rolling circle amplification with decreased fidelity in the presence of MnCl2 and is used to transform a host strain resulting in a mutant library with several random point mutations per kilobase through the entire plasmid. The primary advantage of this method is its simplicity. This protocol does not require the design of specific primers or thermal cycling. The reaction mixture can be used for direct transformation of a host strain. This method allows rapid preparation of randomly mutated plasmid libraries, enabling wider application of random mutagenesis.

  16. 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.

  17. Double-strand break-induced targeted mutagenesis in plants.

    PubMed

    Lyznik, L Alexander; Djukanovic, Vesna; Yang, Meizhu; Jones, Spencer

    2012-01-01

    Double-strand breaks are very potent inducers of DNA recombination. There is no recombination between DNA molecules unless one or two DNA strands are broken. It has become feasible to introduce double-strand breaks at specific chromosomal loci by using dedicated, redesigned endonucleases with altered DNA-binding specificities. Such breaks are mainly repaired by error-prone nonhomologous recombination pathways in somatic cells, thus frequently producing mutations at the preselected chromosomal sites. Although the art and science of reengineering protein properties have been advancing quickly, an empirical validation of new endonucleases in a particular experimental environment is essential for successful targeted mutagenesis experiments. This chapter presents methods that were developed for a comprehensive evaluation of the DNA-binding and DNA-cutting activities of homing endonucleases in maize cells; however, they can be adopted for similar evaluation studies of other endonucleases and other plant species that are amenable for Agrobacterium-mediated transformation. PMID:22351025

  18. 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

  19. 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

  20. 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

  1. 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

  2. Structure-based design of combinatorial mutagenesis libraries.

    PubMed

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

    2015-05-01

    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.

  3. Random mutagenesis by error-prone pol plasmid replication in Escherichia coli.

    PubMed

    Alexander, David L; Lilly, Joshua; Hernandez, Jaime; Romsdahl, Jillian; Troll, Christopher J; Camps, Manel

    2014-01-01

    Directed evolution is an approach that mimics natural evolution in the laboratory with the goal of modifying existing enzymatic activities or of generating new ones. The identification of mutants with desired properties involves the generation of genetic diversity coupled with a functional selection or screen. Genetic diversity can be generated using PCR or using in vivo methods such as chemical mutagenesis or error-prone replication of the desired sequence in a mutator strain. In vivo mutagenesis methods facilitate iterative selection because they do not require cloning, but generally produce a low mutation density with mutations not restricted to specific genes or areas within a gene. For this reason, this approach is typically used to generate new biochemical properties when large numbers of mutants can be screened or selected. Here we describe protocols for an advanced in vivo mutagenesis method that is based on error-prone replication of a ColE1 plasmid bearing the gene of interest. Compared to other in vivo mutagenesis methods, this plasmid-targeted approach allows increased mutation loads and facilitates iterative selection approaches. We also describe the mutation spectrum for this mutagenesis methodology in detail, and, using cycle 3 GFP as a target for mutagenesis, we illustrate the phenotypic diversity that can be generated using our method. In sum, error-prone Pol I replication is a mutagenesis method that is ideally suited for the evolution of new biochemical activities when a functional selection is available.

  4. Functional genomics of the human HDL receptor scavenger receptor BI: An old dog with new tricks

    PubMed Central

    Chadwick, Alexandra C.; Sahoo, Daisy

    2014-01-01

    Purpose of review The athero-protective role of scavenger receptor BI (SR-BI) is primarily attributed to its ability to selectively transfer cholesteryl esters from high density lipoproteins (HDL) to the liver during reverse cholesterol transport (RCT). In this review, we highlight recent findings that reveal the impact of SR-BI on lipid levels and cardiovascular disease in humans. Moreover, additional responsibilities of SR-BI in modulating adrenal and platelet function, as well as female fertility in humans, are discussed. Recent findings Heterozygote carriers of P297S-, S112F- and T175A- mutant SR-BI receptors were identified in patients with high HDL-cholesterol levels. HDL from P297S-SR-BI carriers was unable to mediate macrophage cholesterol efflux, while hepatocytes expressing P297S-SR-BI were unable to mediate the selective uptake of HDL-cholesteryl esters. S112F- and T175A-mutant receptors exhibited similar impaired cholesterol transport functions in vitro. Reduced SR-BI function in P297S carriers was also associated with decreased steroidogenesis and altered platelet function. Further, human population studies identified SCARB1 variants associated with female infertility. Summary Identification of SR-BI variants confirms the key role of this receptor in influencing lipid levels and RCT in humans. Deeper understanding of the contributions of SR-BI to steroidogenesis, platelet function and fertility is required in light of exploration of HDL-raising therapies aimed at reducing cardiovascular risk. PMID:23403740

  5. Improvements in Glucose Sensitivity and Stability of Trichoderma reesei β-Glucosidase Using Site-Directed Mutagenesis

    PubMed Central

    Amano, Yoshihiko

    2016-01-01

    Glucose sensitivity and pH and thermal stabilities of Trichoderma reesei Cel1A (Bgl II) were improved by site-directed mutagenesis of only two amino acid residues (L167W or P172L) at the entrance of the active site. The Cel1A mutant showed high glucose tolerance (50% of inhibitory concentration = 650 mM), glucose stimulation (2.0 fold at 50 mM glucose), and enhanced specific activity (2.4-fold) compared with those of the wild-type Cel1A. Furthermore, the mutant enzyme showed stability at a wide pH range of 4.5–9.0 and possessed high thermal stability up to 50°C with 80% of the residual activities compared with the stability seen at the pH range of 6.5–7.0 and temperatures of up to 40°C in the wild-type Cel1A. Kinetic studies for hydrolysis revealed that the Cel1A mutant was competitively inhibited by glucose at similar levels as the wild-type enzyme. Additionally, the mutant enzyme exhibited substrate inhibition, which gradually disappeared with an increasing glucose concentration. These data suggest that the glucose stimulation was caused by relieve the substrate inhibition in the presence of glucose. To conclude, all the properties improved by the mutagenesis would be great advantages in degradation of cellulosic biomass together with cellulases. PMID:26790148

  6. System-dependent regulations of colour-pattern development: a mutagenesis study of the pale grass blue butterfly

    PubMed Central

    Iwata, Masaki; Hiyama, Atsuki; Otaki, Joji M.

    2013-01-01

    Developmental studies on wing colour patterns have been performed in nymphalid butterflies, but efficient genetic manipulations, including mutagenesis, have not been well established. Here, we have performed mutagenesis experiments in a lycaenid butterfly, the pale grass blue Zizeeria maha, to produce colour-pattern mutants. We fed the P-generation larvae an artificial diet containing the mutagen ethyl methane sulfonate (EMS), and the F1- and F2-generation adults showed various aberrant colour patterns: dorsoventral transformation, anterioposterior background colouration gap, weak contrast, disarrangement of spots, reduction of the size of spots, loss of spots, fusion of spots, and ectopic spots. Among them, the disarrangement, reduction, and loss of spots were likely produced by the coordinated changes of many spots of a single wing around the discal spot in a system-dependent manner, demonstrating the existence of the central symmetry system. The present study revealed multiple genetic regulations for system-dependent and wing-wide colour-pattern determination in lycaenid butterflies. PMID:23917124

  7. 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.

  8. 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

  9. 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

  10. Improvements in Glucose Sensitivity and Stability of Trichoderma reesei β-Glucosidase Using Site-Directed Mutagenesis.

    PubMed

    Guo, Boyang; Amano, Yoshihiko; Nozaki, Kouichi

    2016-01-01

    Glucose sensitivity and pH and thermal stabilities of Trichoderma reesei Cel1A (Bgl II) were improved by site-directed mutagenesis of only two amino acid residues (L167W or P172L) at the entrance of the active site. The Cel1A mutant showed high glucose tolerance (50% of inhibitory concentration = 650 mM), glucose stimulation (2.0 fold at 50 mM glucose), and enhanced specific activity (2.4-fold) compared with those of the wild-type Cel1A. Furthermore, the mutant enzyme showed stability at a wide pH range of 4.5-9.0 and possessed high thermal stability up to 50 °C with 80% of the residual activities compared with the stability seen at the pH range of 6.5-7.0 and temperatures of up to 40 °C in the wild-type Cel1A. Kinetic studies for hydrolysis revealed that the Cel1A mutant was competitively inhibited by glucose at similar levels as the wild-type enzyme. Additionally, the mutant enzyme exhibited substrate inhibition, which gradually disappeared with an increasing glucose concentration. These data suggest that the glucose stimulation was caused by relieve the substrate inhibition in the presence of glucose. To conclude, all the properties improved by the mutagenesis would be great advantages in degradation of cellulosic biomass together with cellulases.

  11. 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

  12. 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

  13. Cryptococcus neoformans virulence gene discovery through insertional mutagenesis.

    PubMed

    Idnurm, Alexander; Reedy, Jennifer L; Nussbaum, Jesse C; Heitman, Joseph

    2004-04-01

    Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to

  14. Genes Necessary for Bacterial Magnetite Biomineralization Identified by Transposon Mutagenesis

    NASA Astrophysics Data System (ADS)

    Nash, C. Z.; Komeili, A.; Newman, D. K.; Kirschvink, J. L.

    2004-12-01

    Magnetic bacteria synthesize nanoscale crystals of magnetite in intracellular, membrane-bounded organelles (magnetosomes). These crystals are preserved in the fossil record at least as far back as the late Neoproterozoic and have been tentatively identified in much older rocks (1). This fossil record may provide deep time calibration points for molecular evolution studies once the genes involved in biologically controlled magnetic mineralization (BCMM) are known. Further, a genetic and biochemical understanding of BCMM will give insight into the depositional environment and biogeochemical cycles in which magnetic bacteria play a role. The BCMM process is not well understood, though proteins have been identified from the magnetosome membrane and genetic manipulation and biochemical characterization of these proteins are underway. Most of the proteins currently thought to be involved are encoded within the mam cluster, a large cluster of genes whose products localize to the magnetosome membrane and are conserved among magnetic bacteria (2). In an effort to identify all of the genes necessary for bacterial BCMM, we undertook a transposon mutagenesis of Magnetospirillum magneticum AMB-1. Non-magnetic mutants (MNMs) were identified by growth in liquid culture followed by a magnetic assay. The insertion site of the transposon was identified two ways. First MNMs were screened with a PCR assay to determine if the transposon had inserted into the mam cluster. Second, the transposon was rescued from the mutant DNA and cloned for sequencing. The majority insertion sites are located within the mam cluster. Insertion sites also occur in operons which have not previously been suspected to be involved in magnetite biomineralization. None of the insertion sites have occurred within genes reported from previous transposon mutagenesis studies of AMB-1 (3, 4). Two of the non-mam cluster insertion sites occur in operons containing genes conserved particularly between MS-1 and MC-1. We

  15. Saturation Mutagenesis of 5S rRNA in Saccharomyces cerevisiae

    PubMed Central

    Smith, Maria W.; Meskauskas, Arturas; Wang, Pinger; Sergiev, Petr V.; Dinman, Jonathan D.

    2001-01-01

    rRNAs are the central players in the reactions catalyzed by ribosomes, and the individual rRNAs are actively involved in different ribosome functions. Our previous demonstration that yeast 5S rRNA mutants (called mof9) can impact translational reading frame maintenance showed an unexpected function for this ubiquitous biomolecule. At the time, however, the highly repetitive nature of the genes encoding rRNAs precluded more detailed genetic and molecular analyses. A new genetic system allows all 5S rRNAs in the cell to be transcribed from a small, easily manipulated plasmid. The system is also amenable for the study of the other rRNAs, and provides an ideal genetic platform for detailed structural and functional studies. Saturation mutagenesis reveals regions of 5S rRNA that are required for cell viability, translational accuracy, and virus propagation. Unexpectedly, very few lethal alleles were identified, demonstrating the resilience of this molecule. Superimposition of genetic phenotypes on a physical map of 5S rRNA reveals the existence of phenotypic clusters of mutants, suggesting that specific regions of 5S rRNA are important for specific functions. Mapping these mutants onto the Haloarcula marismortui large subunit reveals that these clusters occur at important points of physical interaction between 5S rRNA and the different functional centers of the ribosome. Our analyses lead us to propose that one of the major functions of 5S rRNA may be to enhance translational fidelity by acting as a physical transducer of information between all of the different functional centers of the ribosome. PMID:11713264

  16. 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

  17. 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

  18. 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.

  19. 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

  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.

  1. 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

  2. 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

  3. Distribution of Activator (Ac) Throughout the Maize Genome for Use in Regional Mutagenesis

    PubMed Central

    Kolkman, Judith M.; Conrad, Liza J.; Farmer, Phyllis R.; Hardeman, Kristine; Ahern, Kevin R.; Lewis, Paul E.; Sawers, Ruairidh J. H.; Lebejko, Sara; Chomet, Paul; Brutnell, Thomas P.

    2005-01-01

    A collection of Activator (Ac)-containing, near-isogenic W22 inbred lines has been generated for use in regional mutagenesis experiments. Each line is homozygous for a single, precisely positioned Ac element and the Ds reporter, r1-sc:m3. Through classical and molecular genetic techniques, 158 transposed Ac elements (tr-Acs) were distributed throughout the maize genome and 41 were precisely placed on the linkage map utilizing multiple recombinant inbred populations. Several PCR techniques were utilized to amplify DNA fragments flanking tr-Ac insertions up to 8 kb in length. Sequencing and database searches of flanking DNA revealed that the majority of insertions are in hypomethylated, low- or single-copy sequences, indicating an insertion site preference for genic sequences in the genome. However, a number of Ac transposition events were to highly repetitive sequences in the genome. We present evidence that suggests Ac expression is regulated by genomic context resulting in subtle variations in Ac-mediated excision patterns. These tr-Ac lines can be utilized to isolate genes with unknown function, to conduct fine-scale genetic mapping experiments, and to generate novel allelic diversity in applied breeding programs. PMID:15520264

  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. Functional Analysis by Site-Directed Mutagenesis of the NAD+-Reducing Hydrogenase from Ralstonia eutropha

    PubMed Central

    Burgdorf, Tanja; De Lacey, Antonio L.; Friedrich, Bärbel

    2002-01-01

    The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD+ under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H2-oxidizing activity. In one of these isolates (HoxH I64A), H2 binding was impaired. Class II mutants revealed a high D2/H+ exchange rate relative to a low H2-oxidizing activity. A representative (HoxH H16L) displayed D2/H+ exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O2-sensitive growth on hydrogen due to an O2-sensitive SH protein. PMID:12399498

  6. Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection.

    PubMed

    Takasu, Yoko; Kobayashi, Isao; Beumer, Kelly; Uchino, Keiro; Sezutsu, Hideki; Sajwan, Suresh; Carroll, Dana; Tamura, Toshiki; Zurovec, Michal

    2010-10-01

    Targeted mutagenesis is one of the key methods for functional gene analysis. A simplified variant of gene targeting uses direct microinjection of custom-designed Zinc Finger Nuclease (ZFN) mRNAs into Drosophila embryos. To evaluate the applicability of this method to gene targeting in another insect, we mutagenized the Bombyx mori epidermal color marker gene BmBLOS2, which controls the formation of uric acid granules in the larval epidermis. Our results revealed that ZFN mRNA injection is effective to induce somatic, as well as germline, mutations in a targeted gene by non-homologous end joining (NHEJ). The ZFN-induced NHEJ mutations lack end-filling and blunt ligation products, and include mainly 7 bp or longer deletions, as well as single nucleotide insertions. These observations suggest that the B. mori double-strand break repair system relies on microhomologies rather than on a canonical ligase IV-dependent mechanism. The frequency of germline mutants in G(1) was sufficient to be used for gene targeting relying on a screen based solely on molecular methods.

  7. 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

  8. Transplacental teratogenesis and mutagenesis in mouse fetuses treated with cyclophosphamide.

    PubMed

    Porter, A J; Singh, S M

    1988-01-01

    We studied transplacental fetotoxicity, teratogenicity, and mutagenicity in Swiss Webster mice following different doses of cyclophosphamide (CP; 0, 5, 10, 15, or 20 mg/kg), a well-known mutagen/teratogen, on day 12 of gestation. The fetal survival and weight on day 18 of gestation decreased significantly with increasing CP dose (P less than 0.01). The CP-treated fetuses were also dysmorphic (e.g., shortened limbs, digital defects, cleft palate, open eyes, and hydrocephaly) and the percentage of dysmorphology increased with increasing CP doses (P less than 0.01). To evaluate mutagenesis, a separate group of females received 5-bromodeoxyuridine tablet (50-mg) implants on day 12 of gestation and a CP treatment 8 h later. Fetal liver cells were harvested 24 h post-BrdU implant to analyze sister chromatid exchange (SCE) frequency and micronuclei. CP caused a significant increase in the SCEs per fetal liver cell from 3.4 +/- 0.02 (control) to 90.0 +/- 0.04 (20 mg/kg CP) (P less than 0.01). The increasing CP dose was also related to an increase in micronuclei. The data suggest that CP is transplacentally toxic, teratogenic, and mutagenic. Further analyses of the data suggest that the mutagenic effects of CP may in fact contribute indirectly to the CP-related teratogenic effects. Such conclusions are based on path analysis with directional causations associated with SCEs per cell and the dysmorphic features studied.

  9. Retroviral vectors for analysis of viral mutagenesis and recombination.

    PubMed

    Rawson, Jonathan M O; Mansky, Louis M

    2014-09-24

    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.

  10. CRISPR/Cas-mediated targeted mutagenesis in Daphnia magna.

    PubMed

    Nakanishi, Takashi; Kato, Yasuhiko; Matsuura, Tomoaki; Watanabe, Hajime

    2014-01-01

    The water flea Daphnia magna has been used as an animal model in ecology, evolution, and environmental sciences. Thanks to the recent progress in Daphnia genomics, genetic information such as the draft genome sequence and expressed sequence tags (ESTs) is now available. To investigate the relationship between phenotypes and the available genetic information about Daphnia, some gene manipulation methods have been developed. However, a technique to induce targeted mutagenesis into Daphnia genome remains elusive. To overcome this problem, we focused on an emerging genome editing technique mediated by the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system to introduce genomic mutations. In this study, we targeted a functionally conserved regulator of eye development, the eyeless gene in D. magna. When we injected Cas9 mRNAs and eyeless-targeting guide RNAs into eggs, 18-47% of the survived juveniles exhibited abnormal eye morphology. After maturation, up to 8.2% of the adults produced progenies with deformed eyes, which carried mutations in the eyeless loci. These results showed that CRISPR/Cas system could introduce heritable mutations into the endogenous eyeless gene in D. magna. This is the first report of a targeted gene knockout technique in Daphnia and will be useful in uncovering Daphnia gene functions.

  11. 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.

  12. 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

  13. 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.

  14. Overproduction of Clavulanic Acid by UV Mutagenesis of Streptomyces clavuligerus

    PubMed Central

    Korbekandi, Hassan; Darkhal, Parisa; Hojati, Zohreh; Abedi, Daryoush; Hamedi, Javad; Pourhosein, Meraj

    2010-01-01

    Clavulanic acid is produced industrially by fermentation of Streptomyces clavuligerus and researches have increased its production by strain improvement, recombinant DNA technology, and media composition and growth condition optimization. The main objective of this study was to increase the level of clavulanic acid production from Streptomyces clavuligerus (DSM 738), using UV irradiation. After incubation, the spores and aerial mycelia were scraped off the agar plate by a sterile loop. After passing through a cotton wool, the serially diluted spore suspension was spread on GYM- agar containing caffeine. The plates were irradiated with UV light, wrapped in aluminum foil and incubated. The colonies were sub-cultured again to express the mutations. An aliquot of the spore suspension prepared from the resulted culture was poured in GYM agar plates and incubated. The plates were overlaid with nutrient-agar containing penicillin G and Klebsiela pneumoniae, and incubated. The inhibition zone diameter was measured and compared with the wild type colony. Repeating this procedure, the overproducer mutants were selected. Concentration of clavulanic acid was determined by HPLC analysis. It was concluded that secondary metabolites, mainly antibiotics containing clavulanic acid, were produced about 6–7 days after the growth, and concentration of clavulanic acid was increased up to two-folds after UV mutagenesis. PMID:24363725

  15. Multiplex conditional mutagenesis in zebrafish using the CRISPR/Cas system.

    PubMed

    Yin, L; Maddison, L A; Chen, W

    2016-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a powerful tool for genome editing in numerous organisms. However, the system is typically used for gene editing throughout the entire organism. Tissue and temporal specific mutagenesis is often desirable to determine gene function in a specific stage or tissue and to bypass undesired consequences of global mutations. We have developed the CRISPR/Cas system for conditional mutagenesis in transgenic zebrafish using tissue-specific and/or inducible expression of Cas9 and U6-driven expression of sgRNA. To allow mutagenesis of multiple targets, we have isolated four distinct U6 promoters and designed Golden Gate vectors to easily assemble transgenes with multiple sgRNAs. We provide experimental details on the reagents and applications for multiplex conditional mutagenesis in zebrafish. PMID:27443918

  16. Chromosomal aberrations in resident small mammals at a petrochemical waste dump site: a natural model for analysis of environmental mutagenesis. [Peromyscus leucopus; Sigmodon hispidus

    SciTech Connect

    McBee, K.

    1985-01-01

    Small mammals of two species (Peromyscus leucopus and Sigmodon hispidus) were trapped at a locality polluted with a complex mixture of petrochemical waste products, heavy metals, and PCB's, and from two matched, uncontaminated localities. Three cytogenetic techniques were employed to evaluate the use of these resident small mammals as indicators of environmental mutagenesis. Each technique also was assessed for its power of resolution in characterizing the action of environmental mutagens. Standard karyological analysis of flow cytometric analysis clearly indicated significant differences in chromosomal aberrancy between animals collected at the polluted site and the uncontaminated sites. Examination of flow DNA histograms of Peromyscus from the polluted site revealed broadened and flattened G/sub 1/ peaks and increases in CVs (coefficients of variation) for DNA content. CVs in animals from the polluted site consistently fell outside confidence limits set around values from animals collected at the uncontaminated site. These patterns are characteristically seen in laboratory animals challenged with powerful clastogens which suggests that individuals at the polluted site may be experiencing similar clastogenic events. This study demonstrates that small mammals are a feasible test model for evaluating environmental mutagenesis. Evaluation of different cytogenetic techniques suggests that a battery of several assays will provide the most accurate characterization of the action of environmental mutagenesis.

  17. [Dot1 and Set2 Histone Methylases Control the Spontaneous and UV-Induced Mutagenesis Levels in the Saccharomyces cerevisiae Yeasts].

    PubMed

    Kozhina, T N; Evstiukhina, T A; Peshekhonov, V T; Chernenkov, A Yu; Korolev, V G

    2016-03-01

    In the Saccharomyces cerevisiae yeasts, the DOT1 gene product provides methylation of lysine 79 (K79) of hi- stone H3 and the SET2 gene product provides the methylation of lysine 36 (K36) of the same histone. We determined that the dot1 and set2 mutants suppress the UV-induced mutagenesis to an equally high degree. The dot1 mutation demonstrated statistically higher sensitivity to the low doses of MMC than the wild type strain. The analysis of the interaction between the dot1 and rad52 mutations revealed a considerable level of spontaneous cell death in the double dot1 rad52 mutant. We observed strong suppression of the gamma-in- duced mutagenesis in the set2 mutant. We determined that the dot1 and set2 mutations decrease the sponta- neous mutagenesis rate in both single and d ouble mutants. The epistatic interaction between the dot1 and set2 mutations and almost similar sensitivity of the corresponding mutants to the different types of DNA damage allow one to conclude that both genes are involved in the control of the same DNA repair pathways, the ho- mologous-recombination-based and the postreplicative DNA repair.

  18. 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

  19. Application of XeCl308 nm excimer laser radiation to mutagenesis of industrial microorganisms

    NASA Astrophysics Data System (ADS)

    Alifano, P.; Lorusso, A.; Nassisi, V.; Talà, A.; Tredici, S. M.

    (UV) lamps are widely used in mutagenesis-selection protocols. Nevertheless, since the eighties, due to the development of excimer lasers, new frontiers in the study of UV applications have been opened. It has been established that the presence of an intact SOS response system is required for the mutagenic effect of UV254 nm. The exposure to UV254 nm radiation is not mutagenic for Escherichia coli mutants lacking the RecA protein, the regulator of the SOS response. We have recently demonstrated that at variance with the UV254 nm mutagenesis, the UV308 nm mutagenesis by XeCl308 nm excimer laser is RecA-independent. This suggests that the UV308 nm might be mutagenic also in microorganisms naturally lacking the SOS response. In this study, we have developed an innovative mutagenesis protocol based on a homemade XeCl308 nm excimer laser and have demonstrated its efficiency on mutagenesis of Nonomuraea American type culture collection 39727, an industrial strain producing an antibiotic, which is relatively refractory to UV254 nm radiation-induced mutagenesis.

  20. Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis.

    PubMed

    Knäuper, V; Docherty, A J; Smith, B; Tschesche, H; Murphy, G

    1997-03-17

    Analysis of the hinge region of neutrophil collagenase by alanine scanning mutagenesis revealed that this sequence motif has a pronounced effect on the stability and collagenolytic activity of the active enzyme. The mutagenesis of the amino acid residues in the P1' position of the two autoproteolytically cleaved peptide bonds (Leu243 and Ile248) to Ala showed that the mutant enzymes were more resistant to autoproteolysis. However, these mutants were not completely stable and autoproteolysis occurred mainly at the Ala239-Ile240 peptide bond and the half-life of the active enzyme was increased by 50%. In contrast, mutagenesis of Pro247 --> Ala (P1 of the minor cleavage site Pro247-Ile248) lead to increased susceptibility of the enzyme to autoproteolysis. However, when the other P1 position Gly242 was altered to Ala no effect on stability was observed. The analysis of the ability of the mutant active enzymes to hydrolyse 14C-type I collagen was assessed and our results demonstrate that the hinge sequence motif of neutrophil collagenase is important for collagenolytic activity. The alteration of the Gly242-Leu-Ser-Ser-Asn-Pro-Ile-Gln-Pro247 sequence motif to Gly242-Ala-Ala-Ala-Ala-Pro-Ala-Ala-Pro247 showed that the collagenolytic activity was reduced by 68.4%. In addition, mutagenesis of the downstream sequence motif Pro247-Thr-Gly-Pro-Ser-Thr-Pro-Lys-Pro258 to Pro247-Ala-Ala-Pro-Ala-Ala-Pro-Ala-Pro258 had an even more marked effect on the collagenolytic activity, which was reduced by 87.4%. When the Pro residues in the hinge motif (Pro247, Pro250, Pro253 and Pro256) were altered to Ala the collagenolytic activity dropped to 1.5% of the value observed for wild-type enzyme.

  1. Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data

    PubMed Central

    Tripathi, Arti; Gupta, Kritika; Khare, Shruti; Jain, Pankaj C.; Patel, Siddharth; Kumar, Prasanth; Pulianmackal, Ajai J.; Aghera, Nilesh; Varadarajan, Raghavan

    2016-01-01

    Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95pdz3) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo. This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations. PMID:27563054

  2. Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

    PubMed Central

    Caguiat, Jonathan James

    2014-01-01

    Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6Kγ replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host’s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6Kγ replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6Kγ replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. Somatic stem cells and the kinetics of mutagenesis and carcinogenesis

    PubMed Central

    Cairns, John

    2002-01-01

    There is now strong experimental evidence that epithelial stem cells arrange their sister chromatids at mitosis such that the same template DNA strands stay together through successive divisions; DNA labeled with tritiated thymidine in infancy is still present in the stem cells of adult mice even though these cells are incorporating (and later losing) bromodeoxyuridine [Potten, C. S., Owen, G., Booth, D. & Booth, C. (2002) J. Cell Sci.115, 2381–2388]. But a cell that preserves “immortal strands” will avoid the accumulation of replication errors only if it inhibits those pathways for DNA repair that involve potentially error-prone resynthesis of damaged strands, and this appears to be a property of intestinal stem cells because they are extremely sensitive to the lethal effects of agents that damage DNA. It seems that the combination, in the stem cell, of immortal strands and the choice of death rather than error-prone repair makes epithelial stem cell systems resistant to short exposures to DNA-damaging agents, because the stem cell accumulates few if any errors, and any errors made by the daughters are destined to be discarded. This paper discusses these issues and shows that they lead to a model that explains the strange kinetics of mutagenesis and carcinogenesis in adult mammalian tissues. Coincidentally, the model also can explain why cancers arise even though the spontaneous mutation rate of differentiated mammalian cells is not high enough to generate the multiple mutations needed to form a cancer and why loss of nucleotide-excision repair does not significantly increase the frequency of the common internal cancers. PMID:12149477

  9. 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

  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. 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.

  12. 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.

  13. 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

  14. Critical role of arg433 in rat transketolase activity as probed by site-directed mutagenesis.

    PubMed Central

    Soh, Y; Song, B J; Jeng, J; Kallarakal, A T

    1998-01-01

    It has been shown that one arginine per monomer at an unknown position is essential for enzyme activity of the homodimeric transketolase (TK) [Kremer, Egan and Sable (1980) J. Biol. Chem. 255, 2405-2410]. To identify the critical arginine, four highly conserved arginine residues of rat TK (Arg102, Arg350, Arg433 and Arg506) were replaced with alanine by site-directed mutagenesis. Wild-type and mutant TK proteins were produced in Escherichia coli and characterized. The Arg102-->Ala mutant exhibited similar catalytic activity to the wild-type enzyme, whereas Arg350-->Ala, Arg506-->Ala and Arg433-->Ala mutants exhibited 36.7, 37.0 and 6.1% of the wild-type activity respectively. Three recombinant proteins (wild-type, Arg350-->Ala and Arg433-->Ala) were purified to apparent homogeneity using Ni2+-affinity chromatography and further characterized. All these proteins were able to form homodimers (148 kDa), as shown by immunoblot analysis subsequent to non-denaturing gel electrophoresis. The Arg433-->Ala mutant protein was less stable than the wild-type and Arg350-->Ala proteins at 55 degrees C. Kinetic analyses revealed that both Vmax and Km values were markedly affected in the Arg433-->Ala mutant. The Km values for two substrates xylulose 5-phosphate and ribose 5-phosphate were 11.5- and 24.3-fold higher respectively. The kcat/Km values of the Arg433-->Ala mutant for the two substrates were less than 1% of those of the wild-type protein. Molecular modelling of the rat TK revealed that Arg433 of one monomer has three potential hydrogen-bond interactions with the catalytically important highly conserved loop of the other monomer. Thus, our biochemical analyses and modelling data suggest the critical role of the previously uncharacterized Arg433 in TK activity. PMID:9657977

  15. Identification of two new Pmp22 mouse mutants using large-scale mutagenesis and a novel rapid mapping strategy.

    PubMed

    Isaacs, A M; Davies, K E; Hunter, A J; Nolan, P M; Vizor, L; Peters, J; Gale, D G; Kelsell, D P; Latham, I D; Chase, J M; Fisher, E M; Bouzyk, M M; Potter, A; Masih, M; Walsh, F S; Sims, M A; Doncaster, K E; Parsons, C A; Martin, J; Brown, S D; Rastan, S; Spurr, N K; Gray, I C

    2000-07-22

    Mouse mutants have a key role in discerning mammalian gene function and modelling human disease; however, at present mutants exist for only 1-2% of all mouse genes. In order to address this phenotype gap, we have embarked on a genome-wide, phenotype-driven, large-scale N-ethyl-N--nitrosourea (ENU) mutagenesis screen for dominant mutations of clinical and pharmacological interest in the mouse. Here we describe the identification of two similar neurological phenotypes and determination of the underlying mutations using a novel rapid mapping strategy incorporating speed back-crosses and high throughput genotyping. Two mutant mice were identified with marked resting tremor and further characterized using the SHIRPA behavioural and functional assessment protocol. Back-cross animals were generated using in vitro fertilization and genome scans performed utilizing DNA pools derived from multiple mutant mice. Both mutants were mapped to a region on chromosome 11 containing the peripheral myelin protein 22 gene (Pmp22). Sequence analysis revealed novel point mutations in Pmp22 in both lines. The first mutation, H12R, alters the same amino acid as in the severe human peripheral neuropathy Dejerine Sottas syndrome and Y153TER in the other mutant truncates the Pmp22 protein by seven amino acids. Histological analysis of both lines revealed hypo-myelination of peripheral nerves. This is the first report of the generation of a clinically relevant neurological mutant and its rapid genetic characterization from a large-scale mutagenesis screen for dominant phenotypes in the mouse, and validates the use of large-scale screens to generate desired clinical phenotypes in mice. PMID:10915775

  16. MicroRNAs 125a and 455 Repress Lipoprotein-Supported Steroidogenesis by Targeting Scavenger Receptor Class B Type I in Steroidogenic Cells

    PubMed Central

    Hu, Zhigang; Shen, Wen-Jun; Kraemer, Fredric B.

    2012-01-01

    We sought to identify and characterize microRNA (miRNAs) that posttranscriptionally regulate the expression of scavenger receptor class B type I (SR-BI) and SR-BI-linked selective high-density lipoprotein (HDL) cholesteryl ester (CE) transport and steroidogenesis. Four miRNAs (miRNA-125a, miRNA-125b, miRNA-145, and miRNA-455) with a potential to regulate SR-BI were identified in silico and validated by quantitative real-time PCR (qRT-PCR), Western blot analysis, and SR-BI 3′ untranslated region (UTR) reporter assays. In vitro treatment of primary rat granulosa cells and MLTC-1 cells with cyclic AMP (cAMP) or in vivo treatment of rat adrenals with adrenocorticotropic hormone (ACTH) decreased the expression of miRNA-125a, miRNA-125b, and miRNA-455 and reciprocally increased SR-BI expression. Using luciferase constructs containing the 3′ untranslated region of SR-BI combined with miRNA overexpression and mutagenesis, we have provided evidence that steroidogenic SR-BI is a direct target of miRNA-125a and miRNA-455. Moreover, the transfection of Leydig tumor cells with precursor miRNA 125a (pre-miRNA-125a) or pre-miRNA-455 resulted in the suppression of SR-BI at both the transcript and protein levels and reduced selective HDL CE uptake and HDL-stimulated progesterone production. Transfection of liver Hepa 1-6 cells with pre-miRNA-125a significantly reduced SR-BI expression and its selective transport function. In contrast, overexpression of miRNA-145 did not affect SR-BI expression or selective HDL CE uptake mediated by SR-BI in steroidogenic cell lines. These data suggest that a trophic hormone and cAMP inversely regulate the expression of SR-BI and miRNA-125a and miRNA-455 in steroidogenic tissues/cells and that both miRNA-125a and miRNA-455, by targeting steroidogenic SR-BI, negatively regulate selective HDL CE uptake and HDL CE-supported steroid hormone production. PMID:23045399

  17. 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

  18. 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.

  19. Delineation of the complement receptor type 2-C3d complex by site-directed mutagenesis and molecular docking.

    PubMed

    Shaw, Craig D; Storek, Michael J; Young, Kendra A; Kovacs, James M; Thurman, Joshua M; Holers, V Michael; Hannan, Jonathan P

    2010-12-10

    The interactions between the complement receptor type 2 (CR2) and the C3 complement fragments C3d, C3dg, and iC3b are essential for the initiation of a normal immune response. A crystal-derived structure of the two N-terminal short consensus repeat (SCR1-2) domains of CR2 in complex with C3d has previously been elucidated. However, a number of biochemical and biophysical studies targeting both CR2 and C3d appear to be in conflict with these structural data. Previous mutagenesis and heteronuclear NMR spectroscopy studies directed toward the C3d-binding site on CR2 have indicated that the CR2-C3d cocrystal structure may represent an encounter/intermediate or nonphysiological complex. With regard to the CR2-binding site on C3d, mutagenesis studies by Isenman and coworkers [Isenman, D. E., Leung, E., Mackay, J. D., Bagby, S. & van den Elsen, J. M. H. (2010). Mutational analyses reveal that the staphylococcal immune evasion molecule Sbi and complement receptor 2 (CR2) share overlapping contact residues on C3d: Implications for the controversy regarding the CR2/C3d cocrystal structure. J. Immunol. 184, 1946-1955] have implicated an electronegative "concave" surface on C3d in the binding process. This surface is discrete from the CR2-C3d interface identified in the crystal structure. We generated a total of 18 mutations targeting the two (X-ray crystallographic- and mutagenesis-based) proposed CR2 SCR1-2 binding sites on C3d. Using ELISA analyses, we were able to assess binding of mutant forms of C3d to CR2. Mutations directed toward the concave surface of C3d result in substantially compromised CR2 binding. By contrast, targeting the CR2-C3d interface identified in the cocrystal structure and the surrounding area results in significantly lower levels of disruption in binding. Molecular modeling approaches used to investigate disparities between the biochemical data and the X-ray structure of the CR2-C3d cocrystal result in highest-scoring solutions in which CR2 SCR1-2 is

  20. Delineation of the complement receptor type 2-C3d complex by site-directed mutagenesis and molecular docking.

    PubMed

    Shaw, Craig D; Storek, Michael J; Young, Kendra A; Kovacs, James M; Thurman, Joshua M; Holers, V Michael; Hannan, Jonathan P

    2010-12-10

    The interactions between the complement receptor type 2 (CR2) and the C3 complement fragments C3d, C3dg, and iC3b are essential for the initiation of a normal immune response. A crystal-derived structure of the two N-terminal short consensus repeat (SCR1-2) domains of CR2 in complex with C3d has previously been elucidated. However, a number of biochemical and biophysical studies targeting both CR2 and C3d appear to be in conflict with these structural data. Previous mutagenesis and heteronuclear NMR spectroscopy studies directed toward the C3d-binding site on CR2 have indicated that the CR2-C3d cocrystal structure may represent an encounter/intermediate or nonphysiological complex. With regard to the CR2-binding site on C3d, mutagenesis studies by Isenman and coworkers [Isenman, D. E., Leung, E., Mackay, J. D., Bagby, S. & van den Elsen, J. M. H. (2010). Mutational analyses reveal that the staphylococcal immune evasion molecule Sbi and complement receptor 2 (CR2) share overlapping contact residues on C3d: Implications for the controversy regarding the CR2/C3d cocrystal structure. J. Immunol. 184, 1946-1955] have implicated an electronegative "concave" surface on C3d in the binding process. This surface is discrete from the CR2-C3d interface identified in the crystal structure. We generated a total of 18 mutations targeting the two (X-ray crystallographic- and mutagenesis-based) proposed CR2 SCR1-2 binding sites on C3d. Using ELISA analyses, we were able to assess binding of mutant forms of C3d to CR2. Mutations directed toward the concave surface of C3d result in substantially compromised CR2 binding. By contrast, targeting the CR2-C3d interface identified in the cocrystal structure and the surrounding area results in significantly lower levels of disruption in binding. Molecular modeling approaches used to investigate disparities between the biochemical data and the X-ray structure of the CR2-C3d cocrystal result in highest-scoring solutions in which CR2 SCR1-2 is

  1. 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

  2. Exploring the enantioselective mechanism of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by iterative saturation mutagenesis.

    PubMed

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

    2015-04-01

    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.

  3. 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

  4. In vivo evidence for ribavirin-induced mutagenesis of the hepatitis E virus genome

    PubMed Central

    Todt, Daniel; Gisa, Anett; Radonic, Aleksandar; Nitsche, Andreas; Behrendt, Patrick; Suneetha, Pothakamuri Venkata; Pischke, Sven; Bremer, Birgit; Brown, Richard J P; Manns, Michael P; Cornberg, Markus; Bock, C Thomas; Steinmann, Eike; Wedemeyer, Heiner

    2016-01-01

    Objective Hepatitis E virus (HEV) infection can take chronic courses in immunocompromised patients potentially leading to liver cirrhosis and liver failure. Ribavirin (RBV) is currently the only treatment option for many patients, but treatment failure can occur which has been associated with the appearance of a distinct HEV polymerase mutant (G1634R). Here, we performed a detailed analysis of HEV viral intrahost evolution during chronic hepatitis E infections. Design Illumina deep sequencing was performed for the detection of intrahost variation in the HEV genome of chronically infected patients. Novel polymerase mutants were investigated in vitro using state-of-the-art HEV cell culture models. Results Together, these data revealed that (1) viral diversity differed markedly between patients but did not show major intraindividual short-term variations in untreated patients with chronic hepatitis E, (2) RBV therapy was associated with an increase in viral heterogeneity which was reversible when treatment was stopped, (3) the G1634R mutant was detectable as a minor population prior to therapy in patients who subsequently failed to achieve a sustained virological response to RBV therapy and (4) in addition to G1634R further dominant variants in the polymerase region emerged, impacting HEV replication efficiency in vitro. Conclusions In summary, this first investigation of intrahost HEV population evolution indicates that RBV causes HEV mutagenesis in treated patients and that an emergence of distinct mutants within the viral population occurs during RBV therapy. We also suggest that next-generation sequencing could be useful to guide personalised antiviral strategies. PMID:27222534

  5. 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

  6. 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

  7. Large-scale mutagenesis and phenotypic screens for the nervous system and behavior in mice.

    PubMed

    Vitaterna, Martha Hotz; Pinto, Lawrence H; Takahashi, Joseph S

    2006-04-01

    Significant developments have occurred in our understanding of the mammalian genome thanks to informatics, expression profiling and sequencing of the human and rodent genomes. However, although these facets of genomic analysis are being addressed, analysis of in vivo gene function remains a formidable task. Evaluation of the phenotype of mutants provides powerful access to gene function, and this approach is particularly relevant to the nervous system and behavior. Here, we discuss the complementary mouse genetic approaches of gene-driven, targeted mutagenesis and phenotype-driven, chemical mutagenesis. We highlight an NIH-supported large-scale effort to use phenotype-driven mutagenesis screens to identify mouse mutants with neural and behavioral alterations. Such single-gene mutations can then be used for gene identification using positional candidate gene-cloning methods.

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

    PubMed Central

    Varshney, Gaurav Kumar

    2014-01-01

    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

  9. Systematic dissection and trajectory-scanning mutagenesis of the molecular interface that ensures specificity of two-component signaling pathways.

    PubMed

    Capra, Emily J; Perchuk, Barrett S; Lubin, Emma A; Ashenberg, Orr; Skerker, Jeffrey M; Laub, Michael T

    2010-11-24

    Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk.

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

    PubMed

    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

  11. 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

  12. 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

  13. 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.

  14. 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.…

  15. 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...

  16. 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.

  17. DinB upregulation is the sole role of the SOS response in stress-induced mutagenesis in Escherichia coli.

    PubMed

    Galhardo, Rodrigo S; Do, Robert; Yamada, Masami; Friedberg, Errol C; Hastings, P J; Nohmi, Takehiko; Rosenberg, Susan M

    2009-05-01

    Stress-induced mutagenesis is a collection of mechanisms observed in bacterial, yeast, and human cells in which adverse conditions provoke mutagenesis, often under the control of stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e., are stressed. It is therefore important to understand how stress responses increase mutagenesis. In the Escherichia coli Lac assay, stress-induced point mutagenesis requires induction of at least two stress responses: the RpoS-controlled general/starvation stress response and the SOS DNA-damage response, both of which upregulate DinB error-prone DNA polymerase, among other genes required for Lac mutagenesis. We show that upregulation of DinB is the only aspect of the SOS response needed for stress-induced mutagenesis. We constructed two dinB(o(c)) (operator-constitutive) mutants. Both produce SOS-induced levels of DinB constitutively. We find that both dinB(o(c)) alleles fully suppress the phenotype of constitutively SOS-"off" lexA(Ind(-)) mutant cells, restoring normal levels of stress-induced mutagenesis. Thus, dinB is the only SOS gene required at induced levels for stress-induced point mutagenesis. Furthermore, although spontaneous SOS induction has been observed to occur in only a small fraction of cells, upregulation of dinB by the dinB(o(c)) alleles in all cells does not promote a further increase in mutagenesis, implying that SOS induction of DinB, although necessary, is insufficient to differentiate cells into a hypermutable condition. PMID:19270270

  18. Construction of "small-intelligent" focused mutagenesis libraries using well-designed combinatorial degenerate primers.

    PubMed

    Tang, Lixia; Gao, Hui; Zhu, Xuechen; Wang, Xiong; Zhou, Ming; Jiang, Rongxiang

    2012-03-01

    Site-saturation mutagenesis is a powerful tool for protein optimization due to its efficiency and simplicity. A degenerate codon NNN or NNS (K) is often used to encode the 20 standard amino acids, but this will produce redundant codons and cause uneven distribution of amino acids in the constructed library. Here we present a novel "small-intelligent" strategy to construct mutagenesis libraries that have a minimal gene library size without inherent amino acid biases, stop codons, or rare codons of Escherichia coli by coupling well-designed combinatorial degenerate primers with suitable PCR-based mutagenesis methods. The designed primer mixture contains exactly one codon per amino acid and thus allows the construction of small-intelligent mutagenesis libraries with one gene per protein. In addition, the software tool DC-Analyzer was developed to assist in primer design according to the user-defined randomization scheme for library construction. This small-intelligent strategy was successfully applied to the randomization of halohydrin dehalogenases with one or two randomized sites. With the help of DC-Analyzer, the strategy was proven to be as simple as NNS randomization and could serve as a general tool to efficiently randomize target genes at positions of interest.

  19. Gene discovery by chemical mutagenesis and whole-genome sequencing in Dictyostelium.

    PubMed

    Li, Cheng-Lin Frank; Santhanam, Balaji; Webb, Amanda Nicole; Zupan, Blaž; Shaulsky, Gad

    2016-09-01

    Whole-genome sequencing is a useful approach for identification of chemical-induced lesions, but previous applications involved tedious genetic mapping to pinpoint the causative mutations. We propose that saturation mutagenesis under low mutagenic loads, followed by whole-genome sequencing, should allow direct implication of genes by identifying multiple independent alleles of each relevant gene. We tested the hypothesis by performing three genetic screens with chemical mutagenesis in the social soil amoeba Dictyostelium discoideum Through genome sequencing, we successfully identified mutant genes with multiple alleles in near-saturation screens, including resistance to intense illumination and strong suppressors of defects in an allorecognition pathway. We tested the causality of the mutations by comparison to published data and by direct complementation tests, finding both dominant and recessive causative mutations. Therefore, our strategy provides a cost- and time-efficient approach to gene discovery by integrating chemical mutagenesis and whole-genome sequencing. The method should be applicable to many microbial systems, and it is expected to revolutionize the field of functional genomics in Dictyostelium by greatly expanding the mutation spectrum relative to other common mutagenesis methods. PMID:27307293

  20. 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

  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. 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

  3. 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...

  4. 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

  5. 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...

  6. 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

  7. 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.

  8. 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...

  9. 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

  10. 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.

  11. 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

  12. 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.

  13. RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution

    PubMed Central

    Kopsidas, George; Carman, Rachael K; Stutt, Emma L; Raicevic, Anna; Roberts, Anthony S; Siomos, Mary-Anne V; Dobric, Nada; Pontes-Braz, Luisa; Coia, Greg

    2007-01-01

    Background In protein drug development, in vitro molecular optimization or protein maturation can be used to modify protein properties. One basic approach to protein maturation is the introduction of random DNA mutations into the target gene sequence to produce a library of variants that can be screened for the preferred protein properties. Unfortunately, the capability of this approach has been restricted by deficiencies in the methods currently available for random DNA mutagenesis and library generation. Current DNA based methodologies generally suffer from nucleotide substitution bias that preferentially mutate particular base pairs or show significant bias with respect to transitions or transversions. In this report, we describe a novel RNA-based random mutagenesis strategy that utilizes Qβ replicase to manufacture complex mRNA libraries with a mutational spectrum that is close to the ideal. Results We show that Qβ replicase generates all possible base substitutions with an equivalent preference for mutating A/T or G/C bases and with no significant bias for transitions over transversions. To demonstrate the high diversity that can be sampled from a Qβ replicase-generated mRNA library, the approach was used to evolve the binding affinity of a single domain VNAR shark antibody fragment (12Y-2) against malarial apical membrane antigen-1 (AMA-1) via ribosome display. The binding constant (KD) of 12Y-2 was increased by 22-fold following two consecutive but discrete rounds of mutagenesis and selection. The mutagenesis method was also used to alter the substrate specificity of β-lactamase which does not significantly hydrolyse the antibiotic cefotaxime. Two cycles of RNA mutagenesis and selection on increasing concentrations of cefotaxime resulted in mutants with a minimum 10,000-fold increase in resistance, an outcome achieved faster and with fewer overall mutations than in comparable studies using other mutagenesis strategies. Conclusion The RNA based approach

  14. 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

  15. Versatile Vectors for Efficient Mutagenesis of Bradyrhizobium diazoefficiens and Other Alphaproteobacteria

    PubMed Central

    Ledermann, Raphael; Strebel, Silvan; Kampik, Clara

    2016-01-01

    ABSTRACT Analysis of bacterial gene function commonly relies on gene disruption or replacement followed by phenotypic characterization of the resulting mutant strains. Deletion or replacement of targeted regions is commonly achieved via two homologous recombination (HR) events between the bacterial genome and a nonreplicating plasmid carrying DNA fragments flanking the region to be deleted. The counterselection of clones that have integrated the entire plasmid in their genome via a single HR event is crucial in this procedure. Various genetic tools and well-established protocols are available for this type of mutagenesis in model bacteria; however, these methods are not always efficiently applicable in less established systems. Here we describe the construction and application of versatile plasmid vectors pREDSIX and pTETSIX for marker replacement and markerless mutagenesis, respectively. Apart from an array of restriction sites optimized for cloning of GC-rich DNA fragments, the vector backbone contains a constitutively expressed gene for mCherry, enabling the rapid identification of clones originating from single or double HR events by fluorescence-assisted cell sorting (FACS). In parallel, we constructed a series of plasmids from which gene cassettes providing resistance against gentamicin, kanamycin, hygromycin B, streptomycin and spectinomycin, or tetracycline were excised for use with pREDSIX-based marker replacement mutagenesis. In proof-of-concept mutagenesis experiments, we demonstrated the potential for the use of the developed tools for gene deletion mutagenesis in the nitrogen-fixing soybean symbiont Bradyrhizobium diazoefficiens (formerly Bradyrhizobium japonicum) and three additional members of the alphaproteobacteria. IMPORTANCE Mutation and phenotypic analysis are essential to the study of gene function. Efficient mutagenesis protocols and tools are available for many bacterial species, including various model organisms; however, genetic analysis of

  16. 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.

  17. 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.

  18. 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

  19. 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…

  20. 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

  1. 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

  2. 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

  3. 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

  4. Efficient gene-driven germ-line point mutagenesis of C57BL/6J mice

    PubMed Central

    Michaud, Edward J; Culiat, Cymbeline T; Klebig, Mitchell L; Barker, Paul E; Cain, KT; Carpenter, Debra J; Easter, Lori L; Foster, Carmen M; Gardner, Alysyn W; Guo, ZY; Houser, Kay J; Hughes, Lori A; Kerley, Marilyn K; Liu, Zhaowei; Olszewski, Robert E; Pinn, Irina; Shaw, Ginger D; Shinpock, Sarah G; Wymore, Ann M; 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. PMID:16300676

  5. 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.

  6. Extensive mutagenesis of the HSV-1 gB ectodomain reveals remarkable stability of its postfusion form

    PubMed Central

    Vitu, Elvira; Sharma, Sapna; Stampfer, Samuel D.; Heldwein, Ekaterina E.

    2013-01-01

    Viral fusogens mediate the merger of the viral envelope and cellular membrane during viral entry. These proteins share little sequence similarity but all are thought to act by refolding through a series of conformational intermediates from the metastable prefusion form to the stable postfusion form. Crystal structures of both prefusion and postfusion forms have illuminated the conformational pathways of several viral fusogens. By contrast, only the structure of the postfusion form is available for glycoprotein B (gB), the conserved fusogen of herpesviruses. To gain insight into the nature of the fusogenic conformational changes in gB, we used several approaches aimed at engineering the prefusion form of the HSV-1 gB ectodomain, including modifications intended to stabilize the prefusion form and novel mutations aimed at destabilizing the postfusion form. We found that the postfusion conformation of gB is remarkably stable and resistant to perturbations. Several mutations successfully destabilized the gB trimer, identifying regions that are critical for the stability of the postfusion form. Yet, none of the constructs adopted the prefusion conformation. We propose that the soluble ectodomain of gB folds into the postfusion form without first adopting the prefusion intermediate. These results suggest that other regions of gB, including the transmembrane region and the cytoplasmic domain, may be necessary to establish and maintain the metastable prefusion conformation. PMID:23500487

  7. Cysteine mutagenesis reveals alternate proximity between transmembrane domain 2 and hairpin loop 1 of the glutamate transporter EAAT1.

    PubMed

    Zhang, Yunlong; Zhang, Xiuping; Qu, Shaogang

    2014-07-01

    Excitatory amino acid transporter 1 (EAAT1) plays an important role in restricting the neurotoxicity of glutamate. Previous structure-function studies have provided evidence that reentrant helical hairpin loop (HP) 1 has predominant function during the transport cycle. The proposed internal gate HP1 is packed against transmembrane domain (TM) 2 and TM5 in its closed state, and two residues located in TM2 and HP2 of EAAT1 are in close proximity. However, the spatial relationship between TM2 and HP1 during the transport cycle remains unknown. In this study, we used chemical cross-linking of introduced cysteine pair (V96C and S366C) in a cysteine-less version of EAAT1 to assess the proximity of TM2 and HP1. Here, we show that inhibition of transport by copper(II)(1,10-phenanthroline)3 (CuPh) and cadmium ion (Cd(2+)) were observed in the V96C/S366C mutant. Glutamate or potassium significantly protected against the inhibition of transport activity of V96C/S366C by CuPh, while TBOA potentiated the inhibition of transport activity of V96C/S366C by CuPh. We also checked the kinetic parameters of V96C/S366C treated with or without CuPh in the presence of NaCl, NaCl + L-glutamate, NaCl + TBOA, and KCl, respectively. The sensitivity of V96C and S366C to membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium) methanethiosulfonate] was attenuated by glutamate or potassium. TBOA had no effect on the sensitivity of V96C and S366C to MTSET. These data suggest that the spatial relationship between Val-96 of TM2 and Ser-366 of HP1 is altered in the transport cycle.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. Structure analysis and site-directed mutagenesis of defined key residues and motives for pilus-related sortase C1 in group B Streptococcus.

    PubMed

    Cozzi, Roberta; Malito, Enrico; Nuccitelli, Annalisa; D'Onofrio, Mariapina; Martinelli, Manuele; Ferlenghi, Ilaria; Grandi, Guido; Telford, John L; Maione, Domenico; Rinaudo, C Daniela

    2011-06-01

    In group B Streptococcus (GBS), 3 structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular-weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies, and in vivo mutagenesis, we performed a broad characterization of GBS sortase C1 of pilus island 2a. The high-resolution X-ray structure of the enzyme revealed that the active site, into the β-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the "lid." We show that the catalytic triad and the predicted N- and C-terminal transmembrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies, we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild-type protein.

  13. Linker scanner mutagenesis of the Xenopus laevis ribosomal gene promoter.

    PubMed Central

    Reeder, R H; Pennock, D; McStay, B; Roan, J; Tolentino, E; Walker, P

    1987-01-01

    We have assayed a series of linker scanner mutants which cover the Xenopus laevis ribosomal gene promoter at approximately ten base pair intervals. All of these mutations adversely affect promoter activity with the exception of one mutation which stimulates activity. Thus, none are neutral. We show that most of the mutations can be partially rescued by ligating a block of enhancer elements upstream of the promoter. In addition, we have made extracts from liver nuclei which produce DNaseI protection footprints over the promoter. Analysis of both strands reveals a prominent footprinting domain from about -5 to -30. However, lesser changes in the digestion pattern are detected over most of the promoter. Previously published analyses have suggested that this promoter might be composed of three functional domains. The experiments presented here suggest that either 1) the three putative domains are so closely arranged that the boundaries are difficult to discern, or 2) the situation is more complex. Images PMID:3658698

  14. An efficient method for multiple site-directed mutagenesis using type IIs restriction enzymes.

    PubMed

    Zhang, Zhiqiang; Xu, Kun; Xin, Ying; Zhang, Zhiying

    2015-05-01

    Site-directed mutagenesis (SDM) methods are very important in modern molecular biology, biochemistry, and protein engineering. Here, we present a novel SDM method that can be used for multiple mutation generation using type IIs restriction enzymes. This approach is faster and more convenient than the overlap polymerase chain reaction (PCR) method due to its having fewer reaction steps and being cheaper than, but as convenient as, enzymatic assembly. We illustrate the usefulness of our method by introducing three mutations into the bacterial Streptococcus thermophilus Cas9 (bStCas9) gene, converting the humanized S. thermophilus Cas9 (hStCas9) gene into nuclease dead or H847A nickase mutants and generating sunnyTALEN mutagenesis from a wild-type TALEN backbone.

  15. 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

  16. 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

  17. Combinatorial mutagenesis to restrict amino acid usage in an enzyme to a reduced set

    PubMed Central

    Akanuma, Satoshi; Kigawa, Takanori; Yokoyama, Shigeyuki

    2002-01-01

    We developed an effective strategy to restrict the amino acid usage in a relatively large protein to a reduced set with conservation of its in vivo function. The 213-residue Escherichia coli orotate phosphoribosyltransferase was subjected to 22 cycles of segment-wise combinatorial mutagenesis followed by 6 cycles of site-directed random mutagenesis, both coupled with a growth-related phenotype selection. The enzyme eventually tolerated 73 amino acid substitutions: In the final variant, 9 amino acid types (A, D, G, L, P, R, T, V, and Y) occupied 188 positions (88%), and none of 7 amino acid types (C, H, I, M, N, Q, and W) appeared. Therefore, the catalytic function associated with a relatively large protein may be achieved with a subset of the 20 amino acid. The converged sequence also implies simpler constituents for proteins in the early stage of evolution. PMID:12361984

  18. 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

  19. Inhibition of tobacco-induced mutagenesis by eugenol and plant extracts.

    PubMed

    Sukumaran, K; Kuttan, R

    1995-05-01

    Inhibitory effects of eugenol, a compound present in many spices such as cloves, cardamom etc. and the extracts of Anacyclus pyrethrum and Spilanthes calva which are traditionally used in India during the preparation of chewable tobacco, on tobacco-induced mutagenesis were evaluated using Ames Salmonella/microsome assay. Eugenol significantly inhibited (P < 0.001) tobacco-induced mutagenicity at concentrations of 0.5 and 1 mg/plate. Anacyclus pyrethrum extract (1 mg/plate) produced 74.33% inhibition while the extract of Spilanthes calva at 2 mg/plate inhibited tobacco-induced mutagenesis by 86.4%. Eugenol and the plant extracts also inhibited the nitrosation of methylurea in a dose-dependent manner. PMID:7753104

  20. 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/.

  1. Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates.

    PubMed

    Sanchis, Joaquin; Fernández, Layla; Carballeira, J Daniel; Drone, Jullien; Gumulya, Yosephine; Höbenreich, Horst; Kahakeaw, Daniel; Kille, Sabrina; Lohmer, Renate; Peyralans, Jérôme J-P; Podtetenieff, John; Prasad, Shreenath; Soni, Pankaj; Taglieber, Andreas; Wu, Sheng; Zilly, Felipe E; Reetz, Manfred T

    2008-11-01

    Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

  2. 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

  3. Cell survival and shuttle vector mutagenesis induced by ultraviolet A and ultraviolet B radiation in a human cell line.

    PubMed

    Robert, C; Muel, B; Benoit, A; Dubertret, L; Sarasin, A; Stary, A

    1996-04-01

    Although it is known that sunlight is carcinogenic,few molecular data are available concerning the mutagenic effects of ultraviolet (UV) B (290-320 nm) and UVA (320-400 nm) radiation in human cells. To analyze the biologic effects of UVA and UVB, we irradiated the 293 human cell line, derived from adenovirus-transformed human embryonic kidney cells, in which we had stably introduced a shuttle vector harboring the lacZ' bacterial gene as the mutagenesis target. Identical cell survival occurred after UVA doses 700-fold higher than UVB. This comparable to the UVA/UVB ratio that reaches the basal cell layer of the skin after sunlight exposure with UVB sunscreen. The frequency of UVA- and UVB- induced mutations increased with the UV dose as cell survival decreased. At cell survival levels greater than 10%, UVA and UVB induced similar frequencies of mutations in the episomal lacZ gene, whereas for cell survival lower than 10%, UVA induced twice as many mutations as UVB. Sequence analysis of 81 independent lacZ mutants (36 UVA- and 45 UVB-induced) revealed specific characteristics for some UV-induced-mutations, particularly for UVB. Mutations at A/T base pairs were induced more frequently by UVA than by UVB. The UVA-induced mutation spectrum that we have observed in human cells may help help to elucidate the mechanism of skin carcinogenesis. PMID:8618011

  4. 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.

  5. Retroviral expression of the hepatitis B virus x gene promotes liver cell susceptibility to carcinogen-induced site specific mutagenesis.

    PubMed

    Sohn, S; Jaitovitch-Groisman, I; Benlimame, N; Galipeau, J; Batist, G; Alaoui-Jamali, M A

    2000-06-30

    Mutational inactivation of the tumor suppressor gene p53 is common in hepatocellular carcinomas (HCC). AGG to AGT transversion in codon 249 of exon 7 of the p53 gene occurs in over 50% of HCC from endemic regions, where both chronic infection with the hepatitis B virus (HBV) and exposure to carcinogens such as aflatoxin B1 (AFB1) prevail. In this study, we report the effect of the HBV x protein (HBx) on carcinogen-induced cytotoxicity and AGG to AGT mutation in codon 249 of the p53 gene in the human liver cell line CCL13. Expression of HBx, as revealed by its transactivation function, results in enhanced cell susceptibility to cytotoxicity induced by the AFB1 active metabolite, AFB1-8,9-epoxide, and benzo(a)pyrene diol-epoxide. Under similar conditions, expression of HBx promotes apoptosis in a subset of cell population. Exposure to AFB1-8, 9-epoxide alone induces a low frequency of AGG to AGT mutation in codon 249 of the p53 gene, as determined by an allele-specific polymerase chain reaction (AS-PCR) assay. However, expression of HBx enhances the frequency of AFB1-epoxide-induced AGG to AGT mutation compared to control cells. In summary, this study demonstrates that expression of HBx enhances liver cell susceptibility to carcinogen-induced mutagenesis, possibly through alteration of the balance between DNA repair and apoptosis, two cellular defense mechanisms against genotoxic stress. PMID:10856831

  6. Collagen protein abnormalities produced by site-directed mutagenesis of the pro alpha 1(I) gene.

    PubMed

    Bateman, J F; Mascara, T; Cole, W G; Stacey, A; Jaenisch, R

    1989-01-01

    Site-directed mutagenesis of collagen genes offers a powerful new approach for studying structure-function relationships. The construction of engineered mutant collagen genes coding for glycine substitutions and their expression giving rise to the osteogenesis imperfecta type II phenotype in cells and transgenic mice has recently been achieved. This paper further defines the molecular abnormalities of collagen and bone pathology resulting from the expression of the mutant genes.

  7. 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

  8. The mechanism of nucleotide excision repair-mediated UV-induced mutagenesis in nonproliferating cells.

    PubMed

    Kozmin, Stanislav G; Jinks-Robertson, Sue

    2013-03-01

    Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colony-color system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol η translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol ζ-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps.

  9. 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

  10. Identification of putative active-site residues in the DNase domain of colicin E9 by random mutagenesis.

    PubMed

    Garinot-Schneider, C; Pommer, A J; Moore, G R; Kleanthous, C; James, R

    1996-08-01

    We have used random mutagenesis to identify putative active-site residues in the C-terminal cytotoxic endonuclease domain of the bacterial toxin colicin E9. Six single-site mutations in the DNase domain were isolated which destroyed the toxic action of the colicin. DNA sequencing identified the mutations as Gly460Asp, Arg544Gly, Glu548Gly, Thr571Ile, His575Tyr and His579Tyr. All six wild-type residues are highly conserved in the DNase domains of both the E group colicins and the closely related pyocins. Site-directed mutagenesis was then used to substitute the wild-type amino acid residue at each of these positions for an alanine residue in order to distinguish important from unimportant sites. Two of the six alanine-mutant colicins (Gly460Ala and His579Ala) exhibited significant in vivo activity, unlike the original mutation of these residues, and were therefore not characterised further. The Thr571Ala mutant colicin, although not inactive, was significantly less active than the control. The other three alanine mutants (Arg544Ala, Glu548Ala and His575Ala remained completely inactive in the in vivo tests. Each 15 kDa alanine-mutant DNase domain was overexpressed and purified using a tandem-expression strategy which relies on the enzyme being able to bind to the natural inhibitor, Im9. Tryptophan emission spectra of the alanine mutants showed significant alterations in the emission maxima of all but the His575Ala mutant, suggesting changes in the tertiary structure of these mutant proteins. Activity measurements, using the spectrophotometric Kunitz assay, indicated that the Thr571Ala mutant was partially active as an endonuclease but the remaining alanine mutants were all completely inactive. All four mutant proteins, however, retained their ability to bind DNA in a gel shift assay, suggesting the mutations affect catalytic rather than substrate-binding residues. Searching the sequence databases for possible homology to other DNA-binding proteins revealed a

  11. piggyBac-based insertional mutagenesis and enhancer detection as a tool for functional insect genomics.

    PubMed Central

    Horn, Carsten; Offen, Nils; Nystedt, Sverker; Häcker, Udo; Wimmer, Ernst A

    2003-01-01

    Transposon mutagenesis provides a fundamental tool for functional genomics. Here we present a non-species-specific, combined enhancer detection and binary expression system based on the transposable element piggyBac: For the different components of this insertional mutagenesis system, we used widely applicable transposons and distinguishable broad-range transformation markers, which should enable this system to be operational in nonmodel arthropods. In a pilot screen in Drosophila melanogaster, piggyBac mutator elements on the X chromosome were mobilized in males by a Hermes-based jumpstarter element providing piggyBac transposase activity under control of the alpha1-tubulin promoter. As primary reporters in the piggyBac mutator elements, we employed the heterologous transactivators GAL4delta or tTA. To identify larval and adult enhancer detectors, strains carrying UASp-EYFP or TRE-EYFP as secondary reporter elements were used. Tissue-specific enhancer activities were readily observed in the GAL4delta/UASp-based systems, but only rarely in the tTA/TRE system. Novel autosomal insertions were recovered with an average jumping rate of 80%. Of these novel insertions, 3.8% showed homozygous lethality, which was reversible by piggyBac excision. Insertions were found in both coding and noncoding regions of characterized genes and also in noncharacterized and non-P-targeted CG-number genes. This indicates that piggyBac will greatly facilitate the intended saturation mutagenesis in Drosophila. PMID:12618403

  12. Significance of murine retroviral mutagenesis for identification of disease genes in human acute myeloid leukemia.

    PubMed

    Erkeland, Stefan J; Verhaak, Roel G W; Valk, Peter J M; Delwel, Ruud; Löwenberg, Bob; Touw, Ivo P

    2006-01-15

    Retroviral insertion mutagenesis is considered a powerful tool to identify cancer genes in mice, but its significance for human cancer has remained elusive. Moreover, it has recently been debated whether common virus integrations are always a hallmark of tumor cells and contribute to the oncogenic process. Acute myeloid leukemia (AML) is a heterogeneous disease with a variable response to treatment. Recurrent cytogenetic defects and acquired mutations in regulatory genes are associated with AML subtypes and prognosis. Recently, gene expression profiling (GEP) has been applied to further risk stratify AML. Here, we show that mouse leukemia genes identified by retroviral insertion mutagenesis are more frequently differentially expressed in distinct subclasses of adult and pediatric AML than randomly selected genes or genes located more distantly from a virus integration site. The candidate proto-oncogenes showing discriminative expression in primary AML could be placed in regulatory networks mainly involved in signal transduction and transcriptional control. Our data support the validity of retroviral insertion mutagenesis in mice for human disease and indicate that combining these murine screens for potential proto-oncogenes with GEP in human AML may help to identify critical disease genes and novel pathogenetic networks in leukemia.

  13. Lack of mutational hot spots during decitabine-mediated HIV-1 mutagenesis.

    PubMed

    Rawson, Jonathan M O; Landman, Sean R; Reilly, Cavan S; Bonnac, Laurent; Patterson, Steven E; Mansky, Louis M

    2015-11-01

    Decitabine has previously been shown to induce lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1). However, the factors that determine the susceptibilities of individual sequence positions in HIV-1 to decitabine have not yet been defined. To investigate this, we performed Illumina high-throughput sequencing of multiple amplicons prepared from proviral DNA that was recovered from decitabine-treated cells infected with HIV-1. We found that decitabine induced an ≈4.1-fold increase in the total mutation frequency of HIV-1, primarily due to a striking ≈155-fold increase in the G-to-C transversion frequency. Intriguingly, decitabine also led to an ≈29-fold increase in the C-to-G transversion frequency. G-to-C frequencies varied substantially (up to ≈80-fold) depending upon sequence position, but surprisingly, mutational hot spots (defined as upper outliers within the mutation frequency distribution) were not observed. We further found that every single guanine position examined was significantly susceptible to the mutagenic effects of decitabine. Taken together, these observations demonstrate for the first time that decitabine-mediated HIV-1 mutagenesis is promiscuous and occurs in the absence of a clear bias for mutational hot spots. These data imply that decitabine-mediated G-to-C mutagenesis is a highly effective antiviral mechanism for extinguishing HIV-1 infectivity.

  14. Lack of Mutational Hot Spots during Decitabine-Mediated HIV-1 Mutagenesis

    PubMed Central

    Rawson, Jonathan M. O.; Landman, Sean R.; Reilly, Cavan S.; Bonnac, Laurent; Patterson, Steven E.

    2015-01-01

    Decitabine has previously been shown to induce lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1). However, the factors that determine the susceptibilities of individual sequence positions in HIV-1 to decitabine have not yet been defined. To investigate this, we performed Illumina high-throughput sequencing of multiple amplicons prepared from proviral DNA that was recovered from decitabine-treated cells infected with HIV-1. We found that decitabine induced an ≈4.1-fold increase in the total mutation frequency of HIV-1, primarily due to a striking ≈155-fold increase in the G-to-C transversion frequency. Intriguingly, decitabine also led to an ≈29-fold increase in the C-to-G transversion frequency. G-to-C frequencies varied substantially (up to ≈80-fold) depending upon sequence position, but surprisingly, mutational hot spots (defined as upper outliers within the mutation frequency distribution) were not observed. We further found that every single guanine position examined was significantly susceptible to the mutagenic effects of decitabine. Taken together, these observations demonstrate for the first time that decitabine-mediated HIV-1 mutagenesis is promiscuous and occurs in the absence of a clear bias for mutational hot spots. These data imply that decitabine-mediated G-to-C mutagenesis is a highly effective antiviral mechanism for extinguishing HIV-1 infectivity. PMID:26282416

  15. Lethal mutagenesis of foot-and-mouth disease virus involves shifts in sequence space.

    PubMed

    Perales, Celia; Henry, Michel; Domingo, Esteban; Wain-Hobson, Simon; Vartanian, Jean-Pierre

    2011-12-01

    Lethal mutagenesis or virus transition into error catastrophe is an antiviral strategy that aims at extinguishing a virus by increasing the viral mutation rates during replication. The molecular basis of lethal mutagenesis is largely unknown. Previous studies showed that a critical substitution in the foot-and-mouth disease virus (FMDV) polymerase was sufficient to allow the virus to escape extinction through modulation of the transition types induced by the purine nucleoside analogue ribavirin. This substitution was not detected in mutant spectra of FMDV populations that had not replicated in the presence of ribavirin, using standard molecular cloning and nucleotide sequencing. Here we selectively amplify and analyze low-melting-temperature cDNA duplexes copied from FMDV genome populations passaged in the absence or presence of ribovirin Hypermutated genomes with high frequencies of A and U were present in both ribavirin -treated and untreated populations, but the major effect of ribavirin mutagenesis was to accelerate the occurrence of AU-rich mutant clouds during the early replication rounds of the virus. The standard FMDV quasispecies passaged in the absence of ribavirin included the salient transition-modulating, ribavirin resistance mutation, whose frequency increased in populations treated with ribavirin. Thus, even nonmutagenized FMDV quasispecies include a deep, mutationally biased portion of sequence space, in support of the view that the virus replicates close to the error threshold for maintenance of genetic information.

  16. Pseudorabies virus glycoprotein C attachment-proficient revertants isolated through a simple, targeted mutagenesis scheme.

    PubMed

    Rue, Cary A; Ryan, Patrick

    2008-07-01

    Pseudorabies virus (PRV) glycoprotein C (gC) initiates virus attachment to cells by binding to heparan sulfate (HS) proteoglycans. The gC:HS interaction is not essential since gC null mutants still infect; however, they are more easily removed from cells during the initial stages of infection. The expendability of gC has facilitated a genetic mapping of the HS-binding domain, which is composed of three independent heparin-binding domains (HBDs) of six to eight amino acids each. Previous results suggested that at least one of the HBDs (HBD 1) functioned in a context-dependent manner. To define the context better, a reversion analysis was performed in which a defective gC containing a nonfunctional but intact HBD 1 regained HS-binding ability. To increase the reversion frequency, an efficient method for targeted, yet random mutagenesis of the gC gene was developed. The method involves random mutagenesis of a plasmid-borne copy of gC, and highly efficient recombination of the plasmid-borne genes into the virus genome at the site of a double-strand break in the viral gC locus. Revertants were recovered readily, and their gC alleles suggested that HS-binding could be restored by several different amino acid substitutions. This approach should be applicable to targeted mutagenesis of other herpesvirus genes.

  17. Genome-wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus.

    PubMed

    Urbański, Dorian Fabian; Małolepszy, Anna; Stougaard, Jens; Andersen, Stig Uggerhøj

    2012-02-01

    Use of insertion mutants facilitates functional analysis of genes, but it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics in most plant species. The main challenge is developing efficient high-throughput procedures for both mutagenesis and identification of insertion sites. To date, only floral-dip T-DNA transformation of Arabidopsis has produced independent germinal insertions, thereby allowing generation of mutant populations from seeds of single plants. In addition, advances in insertion detection have been hampered by a lack of protocols, including software for automated data analysis, that take full advantage of high-throughput next-generation sequencing. We have addressed these challenges by developing the FSTpoolit protocol and software package, and here we demonstrate its efficacy by detecting 8935 LORE1 insertions in 3744 Lotus japonicus plants. The identified insertions show that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to homogenous gene targeting and exonic insertion preference. As LORE1 transposition occurs in the germline, harvesting seeds from a single founder line and cultivating progeny generates a complete mutant population. This ease of LORE1 mutagenesis, combined with the efficient FSTpoolit protocol, which exploits 2D pooling, Illumina sequencing and automated data analysis, allows highly cost-efficient development of a comprehensive reverse genetic resource.

  18. 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.

  19. Integrated Mimicry of B Cell Antibody Mutagenesis Using Yeast Homologous Recombination

    PubMed Central

    Wittrup, K. Dane

    2014-01-01

    Antibody affinity maturation proceeds in vivo via a combination of point mutations, insertions, deletions, and combinatorial shuffling of light chains or portions of the heavy chain, thereby reducing the probability of trapping in local affinity optima in sequence space. In vivo homologous recombination in yeast can be exploited to mimic the broad spectrum of mutational types deployed by B cells, incorporating both receptor revision and receptor editing together with polymerase-directed point mutagenesis. This method was used to effect a 10,000-fold affinity improvement in an anti-peptide single-chain antibody in three rounds of mutagenesis and screening, and a 1,000-fold affinity improvement in an anti-protein single-chain antibody in a single round. When recombinational mutagenesis (CDR or chain shuffling) was directly compared to error-prone PCR, the recombinational approach yielded greater affinity improvement with substantially reduced divergence from germline sequences, demonstrating an advantage of simultaneously testing a broad range of mutational strategies. PMID:20645027

  20. Integrated mimicry of B cell antibody mutagenesis using yeast homologous recombination.

    PubMed

    Swers, Jeffrey S; Yeung, Yik A; Wittrup, K Dane

    2011-01-01

    Antibody affinity maturation proceeds in vivo via a combination of point mutations, insertions, deletions, and combinatorial shuffling of light chains or portions of the heavy chain, thereby reducing the probability of trapping in local affinity optima in sequence space. In vivo homologous recombination in yeast can be exploited to mimic the broad spectrum of mutational types deployed by B cells, incorporating both receptor revision and receptor editing together with polymerase-directed point mutagenesis. This method was used to effect a 10,000-fold affinity improvement in an anti-peptide single-chain antibody in three rounds of mutagenesis and screening, and a 1,000-fold affinity improvement in an anti-protein single-chain antibody in a single round. When recombinational mutagenesis (CDR or chain shuffling) was directly compared to error-prone PCR, the recombinational approach yielded greater affinity improvement with substantially reduced divergence from germline sequences, demonstrating an advantage of simultaneously testing a broad range of mutational strategies.

  1. 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

  2. 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.

  3. 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

  4. Functional Dissection of an Alternatively Spliced Herpesvirus Gene by Splice Site Mutagenesis

    PubMed Central

    Schommartz, Tim; Loroch, Stefan; Alawi, Malik; Grundhoff, Adam; Sickmann, Albert

    2016-01-01

    ABSTRACT Herpesviruses have large and complex DNA genomes. The largest among the herpesviruses, those of the cytomegaloviruses, include over 170 genes. Although most herpesvirus gene products are expressed from unspliced transcripts, a substantial number of viral transcripts are spliced. Some viral transcripts are subject to alternative splicing, which leads to the expression of several proteins from a single gene. Functional analysis of individual proteins derived from an alternatively spliced gene is difficult, as deletion and nonsense mutagenesis, both common methods used in the generation of viral gene knockout mutants, affect several or all gene products at the same time. Here, we show that individual gene products of an alternatively spliced herpesvirus gene can be inactivated selectively by mutagenesis of the splice donor or acceptor site and by intron deletion or substitution mutagenesis. We used this strategy to dissect the essential M112/113 gene of murine cytomegalovirus (MCMV), which encodes the MCMV Early 1 (E1) proteins. The expression of each of the four E1 protein isoforms was inactivated individually, and the requirement for each isoform in MCMV replication was analyzed in fibroblasts, endothelial cells, and macrophages. We show that the E1 p87 isoform, but not the p33, p36, and p38 isoforms, is essential for viral replication in cell culture. Moreover, the presence of one of the two medium-size isoforms (p36 or p38) and the presence of intron 1, but not its specific sequence, are required for viral replication. This study demonstrates the usefulness of splice site mutagenesis for the functional analysis of alternatively spliced herpesvirus genes. IMPORTANCE Herpesviruses include up to 170 genes in their DNA genomes. The functions of most viral gene products remain poorly defined. The construction of viral gene knockout mutants has thus been an important tool for functional analysis of viral proteins. However, this strategy is of limited use when

  5. 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

  6. 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

  7. 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

  8. Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells.

    PubMed

    Koso, Hideto; Takeda, Haruna; Yew, Christopher Chin Kuan; Ward, Jerrold M; Nariai, Naoki; Ueno, Kazuko; Nagasaki, Masao; Watanabe, Sumiko; Rust, Alistair G; Adams, David J; Copeland, Neal G; Jenkins, Nancy A

    2012-10-30

    Neural stem cells (NSCs) are considered to be the cell of origin of glioblastoma multiforme (GBM). However, the genetic alterations that transform NSCs into glioma-initiating cells remain elusive. Using a unique transposon mutagenesis strategy that mutagenizes NSCs in culture, followed by additional rounds of mutagenesis to generate tumors in vivo, we have identified genes and signaling pathways that can transform NSCs into glioma-initiating cells. Mobilization of Sleeping Beauty transposons in NSCs induced the immortalization of astroglial-like cells, which were then able to generate tumors with characteristics of the mesenchymal subtype of GBM on transplantation, consistent with a potential astroglial origin for mesenchymal GBM. Sequence analysis of transposon insertion sites from tumors and immortalized cells identified more than 200 frequently mutated genes, including human GBM-associated genes, such as Met and Nf1, and made it possible to discriminate between genes that function during astroglial immortalization vs. later stages of tumor development. We also functionally validated five GBM candidate genes using a previously undescribed high-throughput method. Finally, we show that even clonally related tumors derived from the same immortalized line have acquired distinct combinations of genetic alterations during tumor development, suggesting that tumor formation in this model system involves competition among genetically variant cells, which is similar to the Darwinian evolutionary processes now thought to generate many human cancers. This mutagenesis strategy is faster and simpler than conventional transposon screens and can potentially be applied to any tissue stem/progenitor cells that can be grown and differentiated in vitro.

  9. CRISPR/Cas9-mediated targeted mutagenesis in the liverwort Marchantia polymorpha L.

    PubMed

    Sugano, Shigeo S; Shirakawa, Makoto; Takagi, Junpei; Matsuda, Yoriko; Shimada, Tomoo; Hara-Nishimura, Ikuko; Kohchi, Takayuki

    2014-03-01

    Targeted genome modification technologies are key tools for functional genomics. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 system (CRISPR/Cas9) is an emerging technology for targeted genome modification. The CRISPR/Cas9 system consists of a short guide RNA (gRNA), which specifies the target genome sequence, and the Cas9 protein, which has endonuclease activity. The CRISPR/Cas9 system has been applied to model animals and flowering plants, including rice, sorghum, wheat, tobacco and Arabidopsis. Here, we report the application of CRISPR/Cas9 to targeted mutagenesis in the liverwort Marchantia polymorpha L., which has emerged as a model species for studying land plant evolution. The U6 promoter of M. polymorpha was identified and cloned to express the gRNA. The target sequence of the gRNA was designed to disrupt the gene encoding auxin response factor 1 (ARF1) in M. polymorpha. Using Agrobacterium-mediated transformation, we isolated stable mutants in the gametophyte generation of M. polymorpha. CRISPR/Cas9-based site-directed mutagenesis in vivo was achieved using either the Cauliflower mosaic virus 35S or M. polymorpha EF1α promoter to express Cas9. Isolated mutant individuals showing an auxin-resistant phenotype were not chimeric. Moreover, stable mutants were produced by asexual reproduction of T1 plants. Multiple arf1 alleles were easily established using CRIPSR/Cas9-based targeted mutagenesis. Our results provide a rapid and simple approach for molecular genetics in M. polymorpha, and raise the possibility that CRISPR/Cas9 may be applied to a wide variety of plant species.

  10. 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

  11. 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

  12. 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

  13. 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.

  14. Structure-guided alteration of coenzyme specificity of formate dehydrogenase by saturation mutagenesis to enable efficient utilization of NADP+.

    PubMed

    Andreadeli, Aggeliki; Platis, Dimitris; Tishkov, Vladimir; Popov, Vladimir; Labrou, Nikolaos E

    2008-08-01

    Formate dehydrogenase from Candida boidinii (CboFDH) catalyses the oxidation of formate anion to carbon dioxide with concomitant reduction of NAD(+) to NADH. CboFDH is highly specific to NAD(+) and virtually fails to catalyze the reaction with NADP(+). Based on structural information for CboFDH, the loop region between beta-sheet 7 and alpha-helix 10 in the dinucleotide-binding fold was predicted as a principal determinant of coenzyme specificity. Sequence alignment with other formate dehydrogenases revealed two residues (Asp195 and Tyr196) that could account for the observed coenzyme specificity. Positions 195 and 196 were subjected to two rounds of site-saturation mutagenesis and screening and enabled the identification of a double mutant Asp195Gln/Tyr196His, which showed a more than 2 x 10(7)-fold improvement in overall catalytic efficiency with NADP(+) and a more than 900-fold decrease in the efficiency with NAD(+) as cofactors. The results demonstrate that the combined polar interactions and steric factors comprise the main structural determinants responsible for coenzyme specificity. The double mutant Asp195Gln/Tyr196His was tested for practical applicability in a cofactor recycling system composed of cytochrome P450 monooxygenase from Bacillus subtilis, (CYP102A2), NADP(+), formic acid and omega-(p-nitrophenyl)dodecanoic acid (12-pNCA). Using a 1250-fold excess of 12-pNCA over NADP(+) the first order rate constant was determined to be equal to k(obs) = 0.059 +/- 0.004 min(-1).

  15. Evolution of flavone synthase I from parsley flavanone 3beta-hydroxylase by site-directed mutagenesis.

    PubMed

    Gebhardt, Yvonne Helen; Witte, Simone; Steuber, Holger; Matern, Ulrich; Martens, Stefan

    2007-07-01

    Flavanone 3beta-hydroxylase (FHT) and flavone synthase I (FNS I) are 2-oxoglutarate-dependent dioxygenases with 80% sequence identity, which catalyze distinct reactions in flavonoid biosynthesis. However, FNS I has been reported exclusively from a few Apiaceae species, whereas FHTs are more abundant. Domain-swapping experiments joining the N terminus of parsley (Petroselinum crispum) FHT with the C terminus of parsley FNS I and vice versa revealed that the C-terminal portion is not essential for FNS I activity. Sequence alignments identified 26 amino acid substitutions conserved in FHT versus FNS I genes. Homology modeling, based on the related anthocyanidin synthase structure, assigned seven of these amino acids (FHT/FNS I, M106T, I115T, V116I, I131F, D195E, V200I, L215V, and K216R) to the active site. Accordingly, FHT was modified by site-directed mutagenesis, creating mutants encoding from one to seven substitutions, which were expressed in yeast (Saccharomyces cerevisiae) for FNS I and FHT assays. The exchange I131F in combination with either M106T and D195E or L215V and K216R replacements was sufficient to confer some FNS I side activity. Introduction of all seven FNS I substitutions into the FHT sequence, however, caused a nearly complete change in enzyme activity from FHT to FNS I. Both FHT and FNS I were proposed to initially withdraw the beta-face-configured hydrogen from carbon-3 of the naringenin substrate. Our results suggest that the 7-fold substitution affects the orientation of the substrate in the active-site pocket such that this is followed by syn-elimination of hydrogen from carbon-2 (FNS I reaction) rather than the rebound hydroxylation of carbon-3 (FHT reaction).

  16. 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

  17. 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

  18. 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

  19. 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

  20. Molecular dynamics simulation and site-directed mutagenesis of alcohol acyltransferase: a proposed mechanism of catalysis.

    PubMed

    Morales-Quintana, Luis; Nuñez-Tobar, María Ximena; Moya-León, María Alejandra; Herrera, Raúl

    2013-10-28

    Aroma in Vasconcellea pubescens fruit is determined by esters, which are the products of catalysis by alcohol acyltransferase (VpAAT1). VpAAT1 protein structure displayed the conserved HxxxD motif facing the solvent channel in the center of the structure. To gain insight into the role of these catalytic residues, kinetic and site-directed mutagenesis studies were carried out in VpAAT1 protein. Based on dead-end inhibition studies, the kinetic could be described in terms of a ternary complex mechanism with the H166 residue as the catalytic base. Kinetic results showed the lowest Km value for hexanoyl-CoA. Additionally, the most favorable predicted substrate orientation was observed for hexanoyl-CoA, showing a coincidence between kinetic studies and molecular docking analysis. Substitutions H166A, D170A, D170N, and D170E were evaluated in silico. The solvent channel in all mutant structures was lost, showing large differences with the native structure. Molecular docking and molecular dynamics simulations were able to describe unfavored energies for the interaction of the mutant proteins with different alcohols and acyl-CoAs. Additionally, in vitro site-directed mutagenesis of H166 and D170 in VpAAT1 induced a loss of activity, confirming the functional role of both residues for the activity, H166 being directly involved in catalysis.

  1. Control of mammalian cell mutagenesis and differentiation by chemicals which initiate or promote tumor formation

    SciTech Connect

    Jones, C. A.; Huberman, E.

    1980-01-01

    A cell-mediated mutagenesis assay was developed to predict the potential carcinogenic hazard of some environmental chemicals. In this assay, Chinese hamster V79 cells, which are susceptible to mutagenesis, are co-cultivated with cells capable of metabolizing chemical carcinogens. Use of this assay made it possible to demonstrate a relationship between the degree of carcinogenicity and mutagenicity of a series of polycyclic hydrocarbons and nitrosamines and to study the organ specificity exhibited by some chemical carcinogens. However, most short-term in vitro assays are designed to detect mutagenic activity and therefore do not detect tumor promoting agents which are devoid of this activity. By analyzing various markers of terminal differentiation in cultured human melanoma and myeloid leukemia cells, we have established a relationship between the activity of a series of tumor promoting phorbol diesters in the mouse skin and their ability to induce terminal differentiation. We suggest that measuring alterations in the differentiation characteristics of some cultured cells may represent an approach by which environmental tumor promoting agents can be studied and detected.

  2. 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

  3. 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

  4. Human acetyl CoA:arylamine N-acetyltransferase variants generated by random mutagenesis.

    PubMed

    Summerscales, Joanna E; Josephy, P David

    2004-01-01

    Acetyl CoA:arylamine N-acetyltransferase (NAT) enzymes catalyze the N-acetylation of aromatic amines and the O-acetylation of aryl hydroxylamines, reactions that govern the disposition and toxicity of many drugs and carcinogens. The human NAT genes and enzymes NAT1 and NAT2 are highly polymorphic and constitute one of the best studied examples of the genetic control of drug metabolism. Naturally occurring human NAT variants provide limited insight into the relationship between NAT amino acid sequence and enzyme activity. We have shown previously that the expression of recombinant NAT2 in bacterial tester strains results in greatly enhanced sensitivity to mutagenic nitroaromatic compounds (which are reduced to aryl hydroxylamines by bacterial enzymes). We hypothesized that random mutagenesis combined with rapid screening could be used to identify functionally significant amino acid residues in NAT enzymes. Pools of NAT2 variants were generated by polymerase chain reaction-mediated random mutagenesis of the complete coding sequence. Reversion induced by a NAT-dependent mutagen, 3-methyl-2-nitroimidazo[4,5-f]quinoline, was used as the basis for screening these pools to identify variants with altered enzyme activity. Eighteen variants were characterized by quantitative mutagenicity assays and enzyme kinetic measurements. This approach can provide new insight into the biochemistry of enzymes involved in the metabolic activation of mutagens. PMID:14722254

  5. 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.

  6. Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

    PubMed

    Iwakuma, Hidekazu; Koyama, Yoshiyuki; Miyachi, Ayako; Nasukawa, Masashi; Matsumoto, Hitoshi; Yano, Shuntaro; Ogihara, Jun; Kasumi, Takafumi

    2016-01-01

    We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

  7. 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

  8. Mutagenesis of Propionibacterium acnes and analysis of two CAMP factor knock-out mutants.

    PubMed

    Sörensen, Meike; Mak, Tim N; Hurwitz, Robert; Ogilvie, Lesley A; Mollenkopf, Hans J; Meyer, Thomas F; Brüggemann, Holger

    2010-11-01

    P. acnes is a skin commensal that is frequently associated with inflammatory diseases such as acne vulgaris. Despite the availability of the genome sequence functional studies on P. acnes are scarce due to a lack of methods for genetic manipulation of this bacterium. Here we present an insertional mutagenesis approach for the inactivation of specific P. acnes genes. The gene of interest can be disrupted and replaced with an erythromycin-resistance cassette by employing homologous recombination. We used this method to generate knock-out mutants of camp2 (PPA0687) and camp4 (PPA1231), encoding CAMP factor homologs with predicted co-hemolytic activities. The successful inactivation of the two genes was confirmed by PCR and Western blotting experiments using specific anti-CAMP2/CAMP4 sera. The Δcamp2 but not the Δcamp4 mutant exhibited reduced hemolytic activity in the CAMP reaction with sheep erythrocytes, indicating that CAMP2 is the major active co-hemolytic factor of P. acnes. The biological relevance of the CAMP factors remains unclear as disruption of camp2 or camp4 did not significantly alter the transcriptome response of HaCaT cells to P. acnes. The here presented insertional mutagenesis approach will facilitate future studies on P. acnes.

  9. 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.

  10. Single d(ApG)/cis-diamminedichloroplatinum(II) adduct-induced mutagenesis in Escherichia coli

    SciTech Connect

    Burnouf, D.; Fuchs, R.P.P. ); Gauthier, C.; Chottard, J.C. )

    1990-08-01

    The mutation spectrum induced by the widely used antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) showed that cisDDP(d(ApG)) adducts, although they account for only 25% of the lesions formed are {approx}5 times more mutagenic than the major GG adduct. The authors report the construction of vectors bearing a single cisDDP(d(ApG)) lesion and their use in mutagenesis experiments in Escherichia coli. The mutagenic processing of the lesion is found to depend strictly on induction of the SOS system of the bacterial host cells. In SOS-induced cells, mutation frequencies of 1-2% were detected. All these mutations are targeted to the 5{prime} base of the adduct. Single A {yields} T transversions are mainly observed (80%), whereas A {yields} G transitions account for 10% of the total mutations. Tandem base-pair substitutions involving the adenine residue and the thymine residue immediately 5{prime} to the adduct occur at a comparable frequency (10%). No selective loss of the strand bearing the platinum adduct was seen, suggesting that, in vivo, cisDDP(d(ApG)) adducts are not blocking lesions. The high mutation specificity of cisDDP-(d(ApG))-induced mutagenesis is discussed in relation to structural data.

  11. 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

  12. 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

  13. Effects of mutagenesis of murine hepatitis virus nsp1 and nsp14 on replication in culture.

    PubMed

    Eckerle, Lance D; Brockway, Sarah M; Sperry, Steven M; Lu, Xiaotao; Denison, Mark R

    2006-01-01

    For nsp1, the fact that the carboxy-terminal but not the amino-terminal half of the protein can be deleted suggests that there may be specific and distinct domains within the protein or that the entire protein is dispensable but that the RNA encoding the amino-terminal half of nsp1 cannot be deleted. The identification of specific required residues support the conclusion that it is the portion of the protein that is required for replication. The results of mutagenesis of the nsp14 coding region and flanking cleavage sites also provided important new insights into this protein and its requirements. Our previous study raised the question as to the essential nature of nsp14 in replication. The results of this study show that putative active site residues cannot be substituted without loss of replication in culture. Interestingly, mutagenesis of Tyr414 showed that while this residue can tolerate a number of substitutions, it was intolerant of Lysine or deletion. The results suggest that nsp14 is required for replication. However, whatever functions nsp14 serves appear to be retained by noncleaved or partially processed nsp14, since abolition of either the amino-terminal or carboxy-terminal cleavage site allowed recovery of viable virus. PMID:17037504

  14. 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.

  15. The disparity mutagenesis model predicts rescue of living things from catastrophic errors

    PubMed Central

    Furusawa, Mitsuru

    2014-01-01

    In animals including humans, mutation rates per generation exceed a perceived threshold, and excess mutations increase genetic load. Despite this, animals have survived without extinction. This is a perplexing problem for animal and human genetics, arising at the end of the last century, and to date still does not have a fully satisfactory explanation. Shortly after we proposed the disparity theory of evolution in 1992, the disparity mutagenesis model was proposed, which forms the basis for an explanation for an acceleration of evolution and species survival. This model predicts a significant increase of the mutation threshold values if the fidelity difference in replication between the lagging and leading strands is high enough. When applied to biological evolution, the model predicts that living things, including humans, might overcome the lethal effect of accumulated deleterious mutations and be able to survive. Artificially derived mutator strains of microorganisms, in which an enhanced lagging-strand-biased mutagenesis was introduced, showed unexpectedly high adaptability to severe environments. The implications of the striking behaviors shown by these disparity mutators will be discussed in relation to how living things with high mutation rates can avoid the self-defeating risk of excess mutations. PMID:25538731

  16. 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.

  17. Restriction enzyme-free construction of random gene mutagenesis libraries in Escherichia coli.

    PubMed

    Pai, Jen C; Entzminger, Kevin C; Maynard, Jennifer A

    2012-02-15

    Directed evolution relies on both random and site-directed mutagenesis of individual genes and regulatory elements to create variants with altered activity profiles for engineering applications. Central to these experiments is the construction of large libraries of related variants. However, a number of technical hurdles continue to limit routine construction of random mutagenesis libraries in Escherichia coli, in particular, inefficiencies during digestion and ligation steps. Here, we report a restriction enzyme-free approach to library generation using megaprimers termed MegAnneal. Target DNA is first exponentially amplified using error-prone polymerase chain reaction (PCR) and then linearly amplified with a single 3' primer to generate long, randomly mutated, single-stranded megaprimers. These are annealed to single-stranded dUTP-containing template plasmid and extended with T7 polymerase to create a complementary strand, and the resulting termini are ligated with T4 DNA ligase. Using this approach, we are able to reliably generate libraries of approximately 10⁷ colony-forming units (cfu)/μg DNA/transformation in a single day. We have created MegAnneal libraries based on three different single-chain antibodies and identified variants with enhanced expression and ligand-binding affinity. The key advantages of this approach include facile amplification, restriction enzyme-free library generation, and a significantly reduced risk of mutations outside the targeted region and wild-type contamination as compared with current methods.

  18. 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.

  19. Mutagenesis breeding of high echinocandin B producing strain and further titer improvement with culture medium optimization.

    PubMed

    Zou, Shu-Ping; Zhong, Wei; Xia, Chao-Jie; Gu, Ya-Nan; Niu, Kun; Zheng, Yu-Guo; Shen, Yin-Chu

    2015-10-01

    A combination of microbial strain improvement and statistical optimization is investigated to maximize echinocandin B (ECB) production from Aspergillus nidulans ZJB-0817. A classical sequential mutagenesis was studied first by using physical (ultraviolet irradiation at 254 nm) and chemical mutagens (lithium chloride and sodium nitrite). Mutant strain ULN-59 exhibited 2.1-fold increase in ECB production to 1583.1 ± 40.9 mg/L when compared with the parent strain (750.8 ± 32.0 mg/L). This is the first report where mutagenesis is applied in Aspergillus to improve ECB production. Further, fractional factorial design and central composite design were adopted to optimize the culture medium for increasing ECB production by the mutant ULN-59. Results indicated that four culture media including peptone, K2HPO4, mannitol and L-ornithine had significant effects on ECB production. The optimized medium provided another 1.4-fold increase in final ECB concentration to 2285.6 ± 35.6 mg/L compared to the original medium. The results of this study indicated the combined application of a classical mutation and medium optimization can improve effectively ECB production from A. nidulans and could be a promising tool to improve other secondary metabolites production by fungal strains.

  20. 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

  1. 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

  2. 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

  3. Molecular docking and site-directed mutagenesis of a Bacillus thuringiensis chitinase to improve chitinolytic, synergistic lepidopteran-larvicidal and nematicidal activities.

    PubMed

    Ni, Hong; Zeng, Siquan; Qin, Xu; Sun, Xiaowen; Zhang, Shan; Zhao, Xiuyun; Yu, Ziniu; Li, Lin

    2015-01-01

    Bacterial chitinases are useful in the biocontrol of agriculturally important pests and fungal pathogens. However, the utility of naturally occurring bacterial chitinases is often limited by their low enzyme activity. In this study, we constructed mutants of a Bacillus thuringiensis chitinase with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. We first identified a gene from B. thuringiensis YBT-9602 that encodes a chitinase (Chi9602) belonging to glycosyl hydrolase family 18 with conserved substrate-binding and substrate-catalytic motifs. We constructed a structural model of a truncated version of Chi9602 (Chi9602(35-459)) containing the substrate-binding domain using the homologous 1ITX protein of Bacillus circulans as the template. We performed molecular docking analysis of Chi9602(35-459) using di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli. Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several plant-pathogenic fungi were observed.

  4. The role for an invariant aspartic acid in hypoxanthine phosphoribosyltransferases is examined using saturation mutagenesis, functional analysis, and X-ray crystallography.

    PubMed

    Canyuk, B; Focia, P J; Eakin, A E

    2001-03-01

    The role of an invariant aspartic acid (Asp137) in hypoxanthine phosphoribosyltransferases (HPRTs) was examined by site-directed and saturation mutagenesis, functional analysis, and X-ray crystallography using the HPRT from Trypanosoma cruzi. Alanine substitution (D137A) resulted in a 30-fold decrease of k(cat), suggesting that Asp137 participates in catalysis. Saturation mutagenesis was used to generate a library of mutant HPRTs with random substitutions at position 137, and active enzymes were identified by complementation of a bacterial purine auxotroph. Functional analyses of the mutants, including determination of steady-state kinetic parameters and pH-rate dependence, indicate that glutamic acid or glutamine can replace the wild-type aspartate. However, the catalytic efficiency and pH-rate profile for the structural isosteric mutant, D137N, were similar to the D137A mutant. Crystal structures of four of the mutant enzymes were determined in ternary complex with substrate ligands. Structures of the D137E and D137Q mutants reveal potential hydrogen bonds, utilizing several bound water molecules in addition to protein atoms, that position these side chains within hydrogen bond distance of the bound purine analogue, similar in position to the aspartate in the wild-type structure. The crystal structure of the D137N mutant demonstrates that the Asn137 side chain does not form interactions with the purine substrate but instead forms novel interactions that cause the side chain to adopt a nonfunctional rotamer. The results from these structural and functional analyses demonstrate that HPRTs do not require a general base at position 137 for catalysis. Instead, hydrogen bonding sufficiently stabilizes the developing partial positive charge at the N7-atom of the purine substrate in the transition-state to promote catalysis.

  5. Molecular Docking and Site-directed Mutagenesis of a Bacillus thuringiensis Chitinase to Improve Chitinolytic, Synergistic Lepidopteran-larvicidal and Nematicidal Activities

    PubMed Central

    Ni, Hong; Zeng, Siquan; Qin, Xu; Sun, Xiaowen; Zhang, Shan; Zhao, Xiuyun; Yu, Ziniu; Li, Lin

    2015-01-01

    Bacterial chitinases are useful in the biocontrol of agriculturally important pests and fungal pathogens. However, the utility of naturally occurring bacterial chitinases is often limited by their low enzyme activity. In this study, we constructed mutants of a Bacillus thuringiensis chitinase with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. We first identified a gene from B. thuringiensis YBT-9602 that encodes a chitinase (Chi9602) belonging to glycosyl hydrolase family 18 with conserved substrate-binding and substrate-catalytic motifs. We constructed a structural model of a truncated version of Chi9602 (Chi960235-459) containing the substrate-binding domain using the homologous 1ITX protein of Bacillus circulans as the template. We performed molecular docking analysis of Chi960235-459 using di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli. Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several plant-pathogenic fungi were observed. PMID:25678849

  6. 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

  7. 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

  8. 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

    Han, Ruizhi; Liu, Long; Shin, Hyun-Dong; Chen, Rachel R; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-12-01

    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.

  9. Mutagenesis and functional characterization of the RNA and protein components of the toxIN abortive infection and toxin-antitoxin locus of Erwinia.

    PubMed

    Blower, T R; Fineran, P C; Johnson, M J; Toth, I K; Humphreys, D P; Salmond, G P C

    2009-10-01

    Bacteria are constantly challenged by bacteriophage (phage) infection and have developed multiple adaptive resistance mechanisms. These mechanisms include the abortive infection systems, which promote "altruistic suicide" of an infected cell, protecting the clonal population. A cryptic plasmid of Erwinia carotovora subsp. atroseptica, pECA1039, has been shown to encode an abortive infection system. This highly effective system is active across multiple genera of gram-negative bacteria and against a spectrum of phages. Designated ToxIN, this two-component abortive infection system acts as a toxin-antitoxin module. ToxIN is the first member of a new type III class of protein-RNA toxin-antitoxin modules, of which there are multiple homologues cross-genera. We characterized in more detail the abortive infection phenotype of ToxIN using a suite of Erwinia phages and performed mutagenesis of the ToxI and ToxN components. We determined the minimal ToxI RNA sequence in the native operon that is both necessary and sufficient for abortive infection and to counteract the toxicity of ToxN. Furthermore, site-directed mutagenesis of ToxN revealed key conserved amino acids in this defining member of the new group of toxic proteins. The mechanism of phage activation of the ToxIN system was investigated and was shown to have no effect on the levels of the ToxN protein. Finally, evidence of negative autoregulation of the toxIN operon, a common feature of toxin-antitoxin systems, is presented. This work on the components of the ToxIN system suggests that there is very tight toxin regulation prior to suicide activation by incoming phage.

  10. 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.

  11. 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.

  12. Intrinsic and environmental mutagenesis drive diversification and persistence of Pseudomonas aeruginosa in chronic lung infections.

    PubMed

    Rodríguez-Rojas, Alexandro; Oliver, Antonio; Blázquez, Jesús

    2012-01-01

    Pseudomonas aeruginosa is a versatile opportunistic pathogen causing a wide variety of hospital-acquired acute infections in immunocompromised patients as well as chronic respiratory infections in patients suffering from cystic fibrosis or other chronic respiratory diseases. Several traits contribute to its ability to colonize and persist in the lungs of chronically infected patients, including development of high resistance to antimicrobials and hypermutability, biofilm growth, and alginate hyperproduction, or a customized pathogenicity, which may include the loss of classical virulence factors and metabolic changes. Here we argue that a combination of both intrinsic and environmental mutagenesis leads to a high number of mutant variants in the population. The conducive environment then triggers a positive feedback loop leading to adaptation and persistence of P. aeruginosa, rendering these chronic infections almost impossible to eradicate. PMID:22080096

  13. 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

  14. Arenaviruses and Lethal Mutagenesis. Prospects for New Ribavirin-based Interventions

    PubMed Central

    Moreno, Héctor; Grande-Pérez, Ana; Domingo, Esteban; Martín, Verónica

    2012-01-01

    Lymphocytic choriomeningitis virus (LCMV) has contributed to unveil some of the molecular mechanisms of lethal mutagenesis, or loss of virus infectivity due to increased mutation rates. Here we review these developments, and provide additional evidence that ribavirin displays a dual mutagenic and inhibitory activity on LCMV that can be relevant to treatment designs. Using 5-fluorouracil as mutagenic agent and ribavirin either as inhibitor or mutagen, we document an advantage of a sequential inhibitor-mutagen administration over the corresponding combination treatment to achieve a low LCMV load in cell culture. This advantage is accentuated in the concentration range in which ribavirin acts mainly as an inhibitor, rather than as mutagen. This observation reinforces previous theoretical and experimental studies in supporting a sequential inhibitor-mutagen administration as a possible antiviral design. Given recent progress in the development of new inhibitors of arenavirus replication, our results suggest new options of ribavirin-based anti-arenavirus treatments. PMID:23202505

  15. A transposon-based tool for transformation and mutagenesis in trypanosomatid protozoa.

    PubMed

    Damasceno, Jeziel D; Beverley, Stephen M; Tosi, Luiz R O

    2015-01-01

    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.

  16. Linking structure to function: Recent lessons from inositol 1,4,5-trisphosphate receptor mutagenesis.

    PubMed

    Yule, David I; Betzenhauser, Matthew J; Joseph, Suresh K

    2010-06-01

    Great insight has been gained into the structure and function of the inositol 1,4,5 trisphosphate receptor (InsP(3)R) by studies employing mutagenesis of the cDNA encoding the receptor. Notably, early studies using this approach defined the key constituents required for InsP(3) binding in the N-terminus and the membrane spanning regions in the C-terminal domain responsible for channel formation, targeting and function. In this article we evaluate recent studies which have used a similar approach to investigate key residues underlying the in vivo modulation by select regulatory factors. In addition, we review studies defining the structural requirements in the channel domain which comprise the conduction pathway and are suggested to be involved in the gating of the channel.

  17. The hidden side of unstable DNA repeats: mutagenesis at a distance

    PubMed Central

    Shah, Kartik A.; Mirkin, Sergei M.

    2015-01-01

    Structure-prone DNA repeats are common components of genomic DNA in all kingdoms of life. In humans, these repeats are linked to genomic instabilities that result in various hereditary disorders, including many cancers. It has long been known that DNA repeats are not only highly polymorphic in length but can also cause chromosomal fragility and stimulate gross chromosomal rearrangements, i.e. deletions, duplications, inversions, translocations and more complex shuffles. More recently, it has become clear that inherently unstable DNA repeats dramatically elevate mutation rates in surrounding DNA segments and that these mutations can occur upto ten kilobases away from the repetitive tract, a phenomenon we call repeat-induced mutagenesis (RIM). This review describes experimental data that led to the discovery and characterization of RIM and discusses the molecular mechanisms that could account for this phenomenon. PMID:25956860

  18. 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.

  19. A conditional transposon-based insertional mutagenesis screen for hepatocellular carcinoma-associated genes in mice

    PubMed Central

    Keng, Vincent W.; Villanueva, Augusto; Chiang, Derek Y.; Dupuy, Adam J.; Ryan, Barbara J.; Matise, Ilze; Silverstein, Kevin A.T.; Sarver, Aaron; Starr, Timothy K.; Akagi, Keiko; Tessarollo, Lino; Collier, Lara S.; Powers, Scott; Lowe, Scott W.; Jenkins, Nancy A.; Copeland, Neal G.; Llovet, Josep M.; Largaespada, David A.

    2009-01-01

    Here we describe a Sleeping Beauty (SB) transposition system that utilizes a conditional SB transposase allele, which can be activated by Cre recombinase to drive the transposition of a mutagenic transposon in virtually any tissue and control the type of cancer produced. To demonstrate the potential of this system for modeling cancer in mice, we used it to screen for hepatocellular carcinoma (HCC) associated genes in mice by specifically limiting SB transposition to the liver. Among 8,060 non-redundant insertions subsequently cloned from 68 tumor nodules we identified 19 highly significant candidate disease loci, which encode genes like EGFR and MET that are known HCC genes and others like UBE2H that are not strongly implicated in HCC but represent potential new therapeutic targets for treating this neoplasm. With these improvements, transposon-based insertional mutagenesis now offers great potential for better understanding the cancer genome and for identifying new targets for therapeutic development. PMID:19234449

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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

  5. Genetic and physiological modulation of anthracycline-induced mutagenesis in Salmonella typhimurium.

    PubMed

    Cebula, T A

    1986-01-01

    The genotoxic properties of adriamycin and daunomycin, anthracycline antibiotics effective in the treatment of a wide variety of malignancies, were examined in the Salmonella/Ames reverse-mutation test. A novel time- and temperature-dependent phenomenon that potentiates the mutagenicity of these compounds, termed mutational enhancement, is described. The results of congeneric and chemical attenuation studies imply that anthracycline-induced free radicals contribute substantively to the mutagenic potentials of adriamycin and daunomycin. These studies show that adriamycin and daunomycin are not simple intercalative compounds. Rather, anthracycline-induced mutagenesis entails at least two separate but intimately related steps, namely, intercalation within discrete base sequences and the free-radical-mediated events that ensue. Implications of the nonrandom and site-specific action of the anthracyclines are discussed. PMID:3533526

  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. 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

  9. 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

  10. In vivo Elimination of Parental Clones in General and Site-directed Mutagenesis

    PubMed Central

    Holland, Erika G.; Acca, Felicity E.; Belanger, Kristina M.; Bylo, Mary E.; Kay, Brian K.; Weiner, Michael P.; Kiss, Margaret M.

    2015-01-01

    The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5’-CCGCGG-3’ and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 107 from a single transformation and with greater than 90% recombinant clones. PMID:25523926

  11. 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

  12. 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

  13. [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.

  14. 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…

  15. 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...

  16. 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

  17. [Somatic chromosome mutagenesis in residents of Ukraine exposed to ionizing radiation in different periods after the Chernobyl accident].

    PubMed

    Pilinskaia, M A; Dybskiĭ, S S; Shemetun, E V; Dybskaia, E B

    2011-01-01

    The authors summarize results of 25-year selective cytogenetic monitoring of the priority groups in different periods after the Chernobyl accident. The increase in intensity of somatic chromosome mutagenesis in exposed individuals as a result of both targeted and non-targeted radiation-induced cytogenetic effects has been confirmed including delayed, transmissible, hidden chromosome instability and the bystander effect.

  18. 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

  19. 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

  20. 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

  1. Chemical mutagenesis--a promising technique to increase metal concentration and extraction in sunflowers.

    PubMed

    Nehnevajova, Erika; Herzig, Rolf; Federer, Guido; Erismann, Karl-Hans; Schwitzguébel, Jean-Paul

    2007-01-01

    Since most of the metal-hyperaccumulating wild plants only produce very low biomass and many high-yielding crops accumulate only moderate amounts of metals, the current research is mainly focused on overcoming these limitations and the optimization of metal phytoextraction. The main goal of the present study was the improvement of metal concentration and extraction properties of Helianthus annuus L by chemical mutagenesis (the non-GMO approach). Sunflowers--hybrid cultivar Salut and inbred lines-were treated with the chemical mutagen ethyl methanesulfonate (EMS). The effect of chemical mutagenesis on metal concentration in and extraction by new sunflower M1 and M2 mutants was directly assessed on a metal-contaminated field in Raft, Switzerland. Mutants of the M2 generation showed a 2-3 times higher metal shoot concentration than the control plants. The best M2 sunflower "giant mutant" 14/185/04 showed a significantly enhanced metal extraction ability: 7.5 times for Cd, 9.2 times for Zn, and 8.2 times for Pb in aboveground parts, as compared to the control plants. Theoretical calculations for the phytoextraction potential of new sunflower variants note that the best sunflower mutant can produce up to 26 t dry matter per hectare and remove 13.3 kg Zn per hectare and year at the sewage sludge contaminated site of Raft; that is a gain factor of 9 compared to Zn extraction by sunflower controls. Furthermore, the use of sunflower oil and biomass for technical purposes (lubricants, biodiesel, biogas) should produce an additional value and improve the economical balance of phytoextraction.

  2. 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.

  3. Mutagenesis within human FcepsilonRIalpha differentially affects human and murine IgE binding.

    PubMed

    Mackay, Graham A; Hulett, Mark D; Cook, Justin P D; Trist, Halina M; Henry, Alistair J; McDonnell, James M; Beavil, Andrew J; Beavil, Rebecca L; Sutton, Brian J; Hogarth, P Mark; Gould, Hannah J

    2002-02-15

    Soluble fragments of the alpha-chain of FcepsilonRI, the high-affinity receptor for IgE, compete with membrane-bound receptors for IgE and may thus provide a means to combat allergic responses. Mutagenesis within FcepsilonRIalpha is used in this study, in conjunction with the crystal structure of the FcepsilonRIalpha/IgE complex, to define the relative importance of specific residues within human FcepsilonRIalpha for IgE binding. We have also compared the effects of these mutants on binding to both human and mouse IgE, with a view to evaluating the mouse as an appropriate model for the analysis of future agents designed to mimic the human FcepsilonRIalpha and attenuate allergic disease. Three residues within the C-C' region of the FcepsilonRIalpha2 domain and two residues within the alpha2 proximal loops of the alpha1 domain were selected for mutagenesis and tested in binding assays with human and mouse IgE. All three alpha2 mutations (K117D, W130A, and Y131A) reduced the affinity of human IgE binding to different extents, but K117D had a far more pronounced effect on mouse IgE binding, and although Y131A had little effect, W130A modestly enhanced binding to mouse IgE. The mutations in alpha1 (R15A and F17A) diminished binding to both human and mouse IgE, with these effects most likely caused by disruption of the alpha1/alpha2 interface. Our results demonstrate that the effects of mutations in human FcepsilonRIalpha on mouse IgE binding, and hence the inhibitory properties of human receptor-based peptides assayed in rodent models of allergy, may not necessarily reflect their activity in a human IgE-based system.

  4. High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor

    PubMed Central

    Groot-Kormelink, Paul J.; Ferrand, Sandrine; Kelley, Nicholas; Bill, Anke; Freuler, Felix; Imbert, Pierre-Eloi; Marelli, Anthony; Gerwin, Nicole; Sivilotti, Lucia G.; Miraglia, Loren; Orth, Anthony P.; Oakeley, Edward J.; Schopfer, Ulrich; Siehler, Sandra

    2016-01-01

    High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype β1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation. PMID:27649498

  5. 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

  6. High Throughput Random Mutagenesis and Single Molecule Real Time Sequencing of the Muscle Nicotinic Acetylcholine Receptor.

    PubMed

    Groot-Kormelink, Paul J; Ferrand, Sandrine; Kelley, Nicholas; Bill, Anke; Freuler, Felix; Imbert, Pierre-Eloi; Marelli, Anthony; Gerwin, Nicole; Sivilotti, Lucia G; Miraglia, Loren; Orth, Anthony P; Oakeley, Edward J; Schopfer, Ulrich; Siehler, Sandra

    2016-01-01

    High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype β1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation. PMID:27649498

  7. Sleeping Beauty transposon mutagenesis identifies genes that cooperate with mutant Smad4 in gastric cancer development

    PubMed Central

    Takeda, Haruna; Rust, Alistair G.; Ward, Jerrold M.; Yew, Christopher Chin Kuan; Jenkins, Nancy A.; Copeland, Neal G.

    2016-01-01

    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

  8. 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.

  9. 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.

  10. Role of lys100 in human dihydroorotate dehydrogenase: mutagenesis studies and chemical rescue by external amines.

    PubMed

    Jiang, W; Locke, G; Harpel, M R; Copeland, R A; Marcinkeviciene, J

    2000-07-11

    Chemical modification, mutagenesis, chemical rescue, and isotope effect studies are used to identify and probe the roles of several conserved amino acid groups in catalysis by human dihydroorotate dehydrogenase. Time- and pH-dependent inactivation of human dihydroorotate dehydrogenase by trinitrobenzenesulfonate implicates at least one critical lysyl residue in catalysis. Of four highly conserved lysines, only the cognate of Lys255 was previously suggested to have catalytic functionality. We now show that replacement of either Lys184 or Lys186 by mutagenesis does not impact, whereas substitution of Lys100 abolishes, enzymatic activity. However, activity is partially restored to K100C (or K100A) by inclusion of exogenous primary amines in reaction mixtures. This rescued activity saturates with respect to numerous amines and exhibits a steric discrimination reflected in K(d,(amine)) values. For all amines, rescued k(cat) values were only approximately 10% of wild type and independent of amine basicity. K(M) values for dihydroorotate and coenzyme Q(0) were similar to wild type. Thus, exogenous amines (as surrogates for Lys100) apparently complement a chemical, not binding, step(s) of catalysis, which does not entail proton transfer. In support of this postulate, solvent kinetic isotope effect analysis indicates that Lys100 stabilizes developing negative charge on the isoalloxazine ring of flavin mononucleotide during hydride transfer, as has been observed for a number of flavoprotein oxidoreductases. Ser215 of human dihydroarotate dehydrogenase (DHODase) was also studied because of its alignment with the putative active-site base Cys130 of Lactococcus lactisDHODase. Substantial retention of activity by S215C, yet complete loss of activity for S215A, is consistent with Ser215 serving as the active-site base in the human enzyme. PMID:10891080

  11. 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

  12. Trichodiene synthase. Identification of active site residues by site-directed mutagenesis.

    PubMed

    Cane, D E; Shim, J H; Xue, Q; Fitzsimons, B C; Hohn, T M

    1995-02-28

    Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-14C]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat. In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca. 7-8.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7873527

  13. 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

  14. 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

  15. 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

  16. Specificity determinants for autoproteolysis of LexA, a key regulator of bacterial SOS mutagenesis.

    PubMed

    Mo, Charlie Y; Birdwell, L Dillon; Kohli, Rahul M

    2014-05-20

    Bacteria utilize the tightly regulated stress response (SOS) pathway to respond to a variety of genotoxic agents, including antimicrobials. Activation of the SOS response is regulated by a key repressor-protease, LexA, which undergoes autoproteolysis in the setting of stress, resulting in derepression of SOS genes. Remarkably, genetic inactivation of LexA's self-cleavage activity significantly decreases acquired antibiotic resistance in infection models and renders bacteria hypersensitive to traditional antibiotics, suggesting that a mechanistic study of LexA could help inform its viability as a novel target for combating acquired drug resistance. Despite structural insights into LexA, a detailed knowledge of the enzyme's protease specificity is lacking. Here, we employ saturation and positional scanning mutagenesis on LexA's internal cleavage region to analyze >140 mutants and generate a comprehensive specificity profile of LexA from the human pathogen Pseudomonas aeruginosa (LexAPa). We find that the LexAPa active site possesses a unique mode of substrate recognition. Positions P1-P3 prefer small hydrophobic residues that suggest specific contacts with the active site, while positions P5 and P1' show a preference for flexible glycine residues that may facilitate the conformational change that permits autoproteolysis. We further show that stabilizing the β-turn within the cleavage region enhances LexA autoproteolytic activity. Finally, we identify permissive positions flanking the scissile bond (P4 and P2') that are tolerant to extensive mutagenesis. Our studies shed light on the active site architecture of the LexA autoprotease and provide insights that may inform the design of probes of the SOS pathway.

  17. Hepatic Scavenger Receptor BI Protects Against Polymicrobial-induced Sepsis through Promoting LPS Clearance in Mice*

    PubMed Central

    Guo, Ling; Zheng, Zhong; Ai, Junting; Huang, Bin; Li, Xiang-An

    2014-01-01

    Recent studies revealed that scavenger receptor BI (SR-BI or Scarb1) plays a critical protective role in sepsis. However, the mechanisms underlying this protection remain largely unknown. In this study, using Scarb1I179N mice, a mouse model specifically deficient in hepatic SR-BI, we report that hepatic SR-BI protects against cecal ligation and puncture (CLP)-induced sepsis as shown by 75% fatality in Scarb1I179N mice, but only 21% fatality in C57BL/6J control mice. The increase in fatality in Scarb1I179N mice was associated with an exacerbated inflammatory cytokine production. Further study demonstrated that hepatic SR-BI exerts its protection against sepsis through its role in promoting LPS clearance without affecting the inflammatory response in macrophages, the glucocorticoid production in adrenal glands, the leukocyte recruitment to peritoneum or the bacterial clearance in liver. Our findings reveal hepatic SR-BI as a critical protective factor in sepsis and point out that promoting hepatic SR-BI-mediated LPS clearance may provide a therapeutic approach for sepsis. PMID:24719333

  18. Role of Bacillus subtilis DNA Glycosylase MutM in Counteracting Oxidatively Induced DNA Damage and in Stationary-Phase-Associated Mutagenesis

    PubMed Central

    Gómez-Marroquín, Martha; Vidales, Luz E.; Debora, Bernardo N.; Santos-Escobar, Fernando; Obregón-Herrera, Armando; Robleto, Eduardo A.

    2015-01-01

    ABSTRACT Reactive oxygen species (ROS) promote the synthesis of the DNA lesion 8-oxo-G, whose mutagenic effects are counteracted in distinct organisms by the DNA glycosylase MutM. We report here that in Bacillus subtilis, mutM is expressed during the exponential and stationary phases of growth. In agreement with this expression pattern, results of a Western blot analysis confirmed the presence of MutM in both stages of growth. In comparison with cells of a wild-type strain, cells of B. subtilis lacking MutM increased their spontaneous mutation frequency to Rifr and were more sensitive to the ROS promoter agents hydrogen peroxide and 1,1′-dimethyl-4,4′-bipyridinium dichloride (Paraquat). However, despite MutM's proven participation in preventing ROS-induced-DNA damage, the expression of mutM was not induced by hydrogen peroxide, mitomycin C, or NaCl, suggesting that transcription of this gene is not under the control of the RecA, PerR, or σB regulons. Finally, the role of MutM in stationary-phase-associated mutagenesis (SPM) was investigated in the strain B. subtilis YB955 (hisC952 metB5 leuC427). Results revealed that under limiting growth conditions, a mutM knockout strain significantly increased the amount of stationary-phase-associated his, met, and leu revertants produced. In summary, our results support the notion that the absence of MutM promotes mutagenesis that allows nutritionally stressed B. subtilis cells to escape from growth-limiting conditions. IMPORTANCE The present study describes the role played by a DNA repair protein (MutM) in protecting the soil bacterium Bacillus subtilis from the genotoxic effects induced by reactive oxygen species (ROS) promoter agents. Moreover, it reveals that the genetic inactivation of mutM allows nutritionally stressed bacteria to escape from growth-limiting conditions, putatively by a mechanism that involves the accumulation and error-prone processing of oxidized DNA bases. PMID:25825434

  19. Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site-directed mutagenesis

    PubMed Central

    Sherif, Mohammed; Waung, Debbie; Korbeci, Bihter; Mavisakalyan, Valentina; Flick, Robert; Brown, Greg; Abou-Zaid, Mamdouh; Yakunin, Alexander F; Master, Emma R

    2013-01-01

    Summary Multicopper oxidases can act on a broad spectrum of phenolic and non-phenolic compounds. These enzymes include laccases, which are widely distributed in plants and fungi, and were more recently identified in bacteria. Here, we present the results of biochemical and mutational studies of small laccase (SLAC), a multicopper oxidase from Streptomyces coelicolor (SCO6712). In addition to typical laccase substrates, SLAC was tested using phenolic compounds that exhibit antioxidant activity. SLAC showed oxidase activity against 12 of 23 substrates tested, including caffeic acid, ferulic acid, resveratrol, quercetin, morin, kaempferol and myricetin. The kinetic parameters of SLAC were determined for 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), 2,6-dimethoxyphenol, quercetin, morin and myricetin, and maximum reaction rates were observed with myricetin, where kcat and Km values at 60°C were 8.1 (± 0.8) s−1 and 0.9 (± 0.3) mM respectively. SLAC had a broad pH optimum for activity (between pH 4 and 8) and temperature optimum at 60–70°C. It demonstrated remarkable thermostability with a half-life of over 10 h at 80°C and over 7 h at 90°C. Site-directed mutagenesis revealed 17 amino acid residues important for SLAC activity including the 10 His residues involved in copper coordination. Most notably, the Y229A and Y230A mutant proteins showed over 10-fold increase in activity compared with the wild-type SLAC, which was correlated to higher copper incorporation, while kinetic analyses with S929A predicts localization of this residue near the meta-position of aromatic substrates. Funding Information Funding for this research was provided by the Government of Ontario for the project ‘FFABnet: Functionalized Fibre and Biochemicals’ (ORF-RE-05-005), and the Natural Sciences and Engineering Research Council of Canada. PMID:23815400

  20. N-Nitrosocarbaryl-induced mutagenesis in Haemophilus influenzae strains deficient in repair and recombination.

    PubMed

    Beattie, K L

    1975-02-01

    Mutagenesis was studied in repair- and recombination-deficient strains of Haemophilus influenzae after treatment with N-nitrosocarbaryl (NC). Three different strains of H. influenzae carrying mutations affecting excision-repair of UV-induced pyrimidine dimers exhibited normal repair of premutational lesions (as detected by decreased mutation yield resulting from post-treatment DNA synthesis delay) and normal nonreplicative mutation fixation. This indicated that neither of these phenomena are caused by the smae repair mechanism that removes UV-induced pyrimidine dimers from the DNA. The recombination-deficient mutant recI is apparently deficient in the replication-dependent mode of NC-induced mutation fixation. This conclusion is based on the following results: (I) NC-induced mutagenesis is lower in the recI strain than in rec+ cells. (2) Repair of premutational lesions (which depends on the existence of replication-dependent mutation fixation for its detection) was not detected in the recI strain. (3) When nonreplicative mutation fixation and final mutation frequency were measured in the same experiment, about I/4 to I/3 of the final mutation yield could be accounted for by nonreplicative mutation fixation in the rec+ strain, whereas all of the mutation could be accounted for in the recI strain by the nonreplicative mutation fixation. (4) When mutation fixation in strain dna9 recI was followed at the permissive (36 degrees) and nonpermissive (41 degrees) temperatures, it became apparent that in the recI strain replication-dependent mutation fixation occurs at early times, but these newly fixed mutations are unstable and disappear at later times, leaving only the mutations fixed by the nonreplicative process. The recI strain exhibits normal repair of NC-induced single-strand breaks or alkali-labile bonds in the DNA labeled before treatment, but is slow in joining discontinuties present in DNA synthesized after treatment. The results are consistent with the idea that

  1. Improvement of multiple stress tolerance in yeast strain by sequential mutagenesis for enhanced bioethanol production.

    PubMed

    Kumari, Rajni; Pramanik, Krishna

    2012-12-01

    The present work deals with the improvement of multiple stress tolerance in a glucose-xylose co-fermenting hybrid yeast strain RPR39 by sequential mutagenesis using ethyl methane sulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, near and far ultraviolet radiations. The mutants were evaluated for their tolerance to ethanol, temperature and fermentation inhibitors. Among these mutants, mutant RPRT90 exhibited highest tolerance to 10% initial ethanol concentration, 2 g L(-1) furfural and 8 g L(-1) acetic acid. The mutant also showed good growth at high temperature (39-40°C). A study on the combined effect of multiple stresses during fermentation of glucose-xylose mixture (3:1 ratio) was performed using mutant RPRT90. Under the combined effect of thermal (39°C) and inhibitor stress (0.25 g L(-1) vanillin, 0.5 g L(-1) furfural and 4 g L(-1) acetic acid), the mutant produced ethanol with a yield of 0.379 g g(-1), while under combined effect of ethanol (7% v/v) and inhibitor stress the ethanol yield obtained was 0.43 g g(-1). Further, under the synergistic effect of sugar (250 g L(-1)), thermal (39°C), ethanol (7% v/v) and inhibitors stress, the strain produced a maximum of 47.93 g L(-1) ethanol by utilizing 162.42 g L(-1) of glucose-xylose mixture giving an ethanol yield of 0.295 g g(-1) and productivity of 0.57 g L(-1) h(-1). Under same condition the fusant RPR39 produced a maximum of 30.0 g L(-1) ethanol giving a yield and productivity of 0.21 g g(-1) and 0.42 g L(-1) h(-1) respectively. The molecular characterization of mutant showed considerable difference in its genetic profile from hybrid RPR39. Thus, sequential mutagenesis was found to be effective to improve the stress tolerance properties in yeast.

  2. Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice[S

    PubMed Central

    Thacker, Seth G.; Rousset, Xavier; Esmail, Safiya; Zarzour, Abdalrahman; Jin, Xueting; Collins, Heidi L.; Sampson, Maureen; Stonik, John; Demosky, Stephen; Malide, Daniela A.; Freeman, Lita; Vaisman, Boris L.; Kruth, Howard S.; Adelman, Steven J.; Remaley, Alan T.

    2015-01-01

    LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(−/−)] mice, which have a secondary defect in cholesterol esterification. Scarab(−/−)×LCAT-null [Lcat(−/−)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(−/−)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(−/−)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(−/−)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(−/−) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles. PMID:25964513

  3. Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice.

    PubMed

    Thacker, Seth G; Rousset, Xavier; Esmail, Safiya; Zarzour, Abdalrahman; Jin, Xueting; Collins, Heidi L; Sampson, Maureen; Stonik, John; Demosky, Stephen; Malide, Daniela A; Freeman, Lita; Vaisman, Boris L; Kruth, Howard S; Adelman, Steven J; Remaley, Alan T

    2015-07-01

    LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(-/-)] mice, which have a secondary defect in cholesterol esterification. Scarab(-/-)×LCAT-null [Lcat(-/-)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(-/-)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(-/-)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(-/-)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(-/-) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles. PMID:25964513

  4. 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

  5. Random mutagenesis and media optimisation for hyperproduction of cellulase from Bacillus species using proximally analysed Saccharum spontaneum in submerged fermentation.

    PubMed

    Abdullah, Roheena; Zafar, Wajeeha; Nadeem, Muhammad; Iqtedar, Mehwish; Naz, Shagufta; Syed, Quratulain; Butt, Zahid Ali

    2015-01-01

    This study deals with the isolation of novel mutant of Bacillus and optimisation of media for the hyperproduction of cellulase. Cellulase-producing Bacillus PC-BC6 was subjected to physical and chemical mutagenesis to enhance the cellulolytic potential. Later, mutagenesis isolates were screened both qualitatively and quantitatively. Among all the tested isolates, Bacillus N3 yielded maximum (CMCase 1250 IU/mL/min and FPase 629 IU/mL/min) activity. The Bacillus N3 strain exhibited 1.7-fold more enzyme production as compared with the parental strain. Proximate analysis of untreated and pretreated Saccharum spontaneum was carried out to improve cellulase production. Three different media were tested for the production of cellulase, among which M2 medium containing MgSO4, pretreated S. spontaneum, K2HPO4, (NH4)2SO4 and peptone was found to be the best for maximum enzyme production by mutant Bacillus N3.

  6. 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

  7. Site-directed mutagenesis of the glycine-rich loop of death associated protein kinase (DAPK) identifies it as a key structure for catalytic activity.

    PubMed

    McNamara, Laurie K; Brunzelle, Joseph S; Schavocky, James P; Watterson, D Martin; Grum-Tokars, Valerie

    2011-05-01

    Death associated protein kinase (DAPK) is a calmodulin (CaM)-regulated protein kinase that is a therapeutic target for central nervous system (CNS) disorders. We report here the results of studies that test the hypothesis of McNamara et al. (2009) that conformational selection in DAPK's glycine-rich region is key for catalytic activity. The hypothesis was tested by site-directed mutagenesis of glutamine-23 (Q23) in the middle of this loop. The glycine-rich loop exhibits localized differences in structure among DAPK conformations that correlate with different stages of the catalytic cycle. Changing the Q23 to a Valine (V23), found at the corresponding position in another CaM regulated protein kinase, results in a reduced catalytic efficiency. High resolution X-ray crystal structures of various conformations of the Q23V mutant DAPK and their superimposition with the corresponding conformations from wild type catalytic domain reveal localized changes in the glycine-rich region. The effect of the mutation on DAPK catalytic activity and the finding of only localized changes in the DAPK structure provide experimental evidence implicating conformational selection in this domain with activity. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

  8. 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

  9. Sequence-structure-function relationships of a tRNA (m7G46) methyltransferase studied by homology modeling and site-directed mutagenesis.

    PubMed

    Purta, Elzbieta; van Vliet, Françoise; Tricot, Catherine; De Bie, Lara G; Feder, Marcin; Skowronek, Krzysztof; Droogmans, Louis; Bujnicki, Janusz M

    2005-05-15

    The Escherichia coli TrmB protein and its Saccharomyces cerevisiae ortholog Trm8p catalyze the S-adenosyl-L-methionine-dependent formation of 7-methylguanosine at position 46 (m7G46) in tRNA. To learn more about the sequence-structure-function relationships of these enzymes we carried out a thorough bioinformatics analysis of the tRNA:m7G methyltransferase (MTase) family to predict sequence regions and individual amino acid residues that may be important for the interactions between the MTase and the tRNA substrate, in particular the target guanosine 46. We used site-directed mutagenesis to construct a series of alanine substitutions and tested the activity of the mutants to elucidate the catalytic and tRNA-recognition mechanism of TrmB. The functional analysis of the mutants, together with the homology model of the TrmB structure and the results of the phylogenetic analysis, revealed the crucial residues for the formation of the substrate-binding site and the catalytic center in tRNA:m7G MTases.

  10. Cloning and expression of the carboxypeptidase gene from Aspergillus saitoi and determination of the catalytic residues by site-directed mutagenesis.

    PubMed Central

    Chiba, Y; Midorikawa, T; Ichishima, E

    1995-01-01

    Carboxypeptidase from Aspergillus saitoi removes acidic, neutral and basic amino acids as well as proline from the C-terminal position at pH 2-5. cpdS, a cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. Analysis of the 1816-nucleotide sequence revealed a single open reading frame coding for 523 amino acids. When A. saitoi carboxypeptidase cDNA was expressed in yeast cells, carboxypeptidase activity was detected in the cell extract and was immunostained with a 72 kDa protein with polyclonal anti-(A. saitoi carboxypeptidase) serum. The recombinant enzyme treated with glycopeptidase F migrated with an apparent molecular mass of 60 kDa on SDS/PAGE, which was the same as that of the de-N-glycosylated carboxypeptidase from A. saitoi. Site-directed mutagenesis of the cpdS indicated that Ser-153, Asp-357 and His-436 residues were essential for the enzymic catalysis. It can be concluded that A. saitoi carboxypeptidase has a catalytic triad comprising Asp-His-Ser and is a member of serine carboxypeptidase family (EC 3.4.16.1). Images Figure 3 Figure 4 Figure 5 PMID:7772020

  11. Site-Directed Mutagenesis and Structural Studies Suggest that the Germination Protease, GPR, in Spores of Bacillus Species Is an Atypical Aspartic Acid Protease

    PubMed Central

    Carroll, Thomas M.; Setlow, Peter

    2005-01-01

    Germination protease (GPR) initiates the degradation of small, acid-soluble spore proteins (SASP) during germination of spores of Bacillus and Clostridium species. The GPR amino acid sequence is not homologous to members of the major protease families, and previous work has not identified residues involved in GPR catalysis. The current work has focused on identifying catalytically essential amino acids by mutagenesis of Bacillus megaterium gpr. A residue was selected for alteration if it (i) was conserved among spore-forming bacteria, (ii) was a potential nucleophile, and (iii) had not been ruled out as inessential for catalysis. GPR variants were overexpressed in Escherichia coli, and the active form (P41) was assayed for activity against SASP and the zymogen form (P46) was assayed for the ability to autoprocess to P41. Variants inactive against SASP and unable to autoprocess were analyzed by circular dichroism spectroscopy and multiangle laser light scattering to determine whether the variant's inactivity was due to loss of secondary or quaternary structure, respectively. Variation of D127 and D193, but no other residues, resulted in inactive P46 and P41, while variants of each form were well structured and tetrameric, suggesting that D127 and D193 are essential for activity and autoprocessing. Mapping these two aspartate residues and a highly conserved lysine onto the B. megaterium P46 crystal structure revealed a striking similarity to the catalytic residues and propeptide lysine of aspartic acid proteases. These data indicate that GPR is an atypical aspartic acid protease. PMID:16199582

  12. Alanine-scanning mutagenesis defines a conserved energetic hotspot in the CaVα1 AID-CaVβ interaction site that is critical for channel modulation

    PubMed Central

    Van Petegem, Filip; Duderstadt, Karl E.; Clark, Kimberly A.; Wang, Michelle; Minor, Daniel L.

    2011-01-01

    Voltage-gated calcium channels (CaVs) are large, multisubunit complexes that control cellular calcium entry. CaV pore-forming (CaVα1) and cytoplasmic (CaVβ) subunits associate through a high-affinity interaction between the CaVα1 α-interaction domain (AID) and CaVβ α-binding pocket (ABP). Here, we analyze AID-ABP interaction thermodynamics using isothermal titration calorimetry (ITC). We find that commensurate with their strong sequence similarity, all CaV1 and CaV2 AID peptides bind CaVβ with similar nanomolar affinities. Although the AID-ABP interface encompasses twenty-four sidechains, alanine-scanning mutagenesis reveals that the binding energy is focused in two complementary hotspots comprising four deeply-conserved residues. Electrophysiological experiments show that hotspot interaction disruption prevents trafficking and functional modulation of CaV1.2 by CaVβ. Together, the data support the primacy of the AID-ABP interface for CaVα1-CaVβ association, underscore the idea that hotspots dominate protein-protein interaction affinities, and uncover a target for strategies to control cellular excitability by blocking CaVα1-CaVβ complex formation. PMID:18275819

  13. Identification of the roles of individual amino acid residues of the helix E of the major antenna of photosystem II (LHCII) by alanine scanning mutagenesis.

    PubMed

    Liu, Cheng; Rao, Yan; Zhang, Lei; Yang, Chunhong

    2014-10-01

    The functions of the helix E (W97-F105), an amphiphilic lumenal 310 helix of the major antenna of photosystem II (LHCII), are still unidentified. To elucidate the roles of individual amino acid residue of the helix E, alanine scanning mutagenesis has been performed to mutate every residue of this domain to alanine. The influence of every alanine substitution on the structure and function of LHCII has been investigated biochemically and spectroscopically. The results show that all mutations have little impact on the pigment binding and configuration. However, many mutants presented decreased thermo- or photo-stability compared with the wild type, highlighting the significance of this helix to the stability of LHCII. The most critical residue for stability is W97. The mutant W97A yielded very fragile trimeric pigment protein complexes. The structural analysis revealed that the hydrogen bonding and aromatic interactions between W97, F195, F194 and a water molecule contributed greatly to the stability of LHCII. Moreover, Q103A and F105A have been identified to be able to reinforce the tendency of aggregation in vitro. The structural analysis suggested that the enhancement in aggregation formation for Q103A and F105A might be attributed to the changing hydrophobicity of the region.

  14. Transcription-dependent cytosine deamination is a novel mechanism in ultraviolet light-induced mutagenesis.

    PubMed

    Hendriks, Giel; Calléja, Fabienne; Besaratinia, Ahmad; Vrieling, Harry; Pfeifer, Gerd P; Mullenders, Leon H F; Jansen, Jacob G; de Wind, Niels

    2010-01-26

    Skin cancer is the most ubiquitous cancer type in the Caucasian population, and its incidence is increasing rapidly [1]. Transcribed proliferation-related genes in dermal stem cells are targets for the induction of ultraviolet light (UV)-induced mutations that drive carcinogenesis. We have recently found that transcription of a gene increases its mutability by UV in mammalian stem cells, suggesting a role of transcription in skin carcinogenesis [2]. Here we show that transcription-associated UV-induced nucleotide substitutions are caused by increased deamination of cytosines to uracil within photolesions at the transcribed strand, presumably at sites of stalled transcription complexes. Additionally, via an independent mechanism, transcription of UV-damaged DNA induces the generation of intragenic deletions. We demonstrate that transcription-coupled nucleotide excision repair (TC-NER) provides protection against both classes of transcription-associated mutagenesis. Combined, these results unveil the existence of two mutagenic pathways operating specifically at the transcribed DNA strand of active genes. Moreover, these results uncover a novel role for TC-NER in the suppression of UV-induced genome aberrations and provide a rationale for the efficient induction of apoptosis by stalled transcription complexes. PMID:20045328

  15. Analysis of benzo[a]pyrene diolepoxide mutagenesis in a mammalian in vitro DNA replication system

    SciTech Connect

    Vasunia, K.; Cheng, L.; Carty, M.

    1995-11-01

    Chemicals that interact with DNA and cause mutations, thereby activating protooncogenes or inactivating tumor suppressor genes, are thought to initiate the process of carcinogenesis. To elucidate the molecular mechanisms involved in mutagenesis of bulky adducts, we used an in vitro DNA replication system. An SV40-based shuttle vector, PZ189, was treated with anti-benzo[a]pyrene-7,8- dihydrodiol-9,10-epoxide (BPDE) and then replicated in vitro using hypotonic extracts of human HeLa cells. The replication efficiency was monitored by the incorporation of a radiolabelled nucleotide into DNA. The products of the replication reaction were then digested with Dpn-1 to inactivate the unreplicated plasmid and the mutation frequency was evaluated by transfection into E. coli MBM7070. Our results show that BPDE-damaged plasmids undergo replication in the in vitro system. The efficiency of replication and the mutant frequency is dose-dependent, such that the replication efficiency decreases and mutation frequency increases with increasing BPDE dose to the plasmid. This study further validates the in vitro DNA replication system by demonstrating the mutagenicity of bulky adducts of BPDE.

  16. 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

  17. 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.

  18. Genomic Access to Monarch Migration Using TALEN and CRISPR/Cas9-Mediated Targeted Mutagenesis

    PubMed Central

    Markert, Matthew J.; Zhang, Ying; Enuameh, Metewo S.; Reppert, Steven M.; Wolfe, Scot A.; Merlin, Christine

    2016-01-01

    The eastern North American monarch butterfly, Danaus plexippus, is an emerging model system to study the neural, molecular, and genetic basis of animal long-distance migration and animal clockwork mechanisms. While genomic studies have provided new insight into migration-associated and circadian clock genes, the general lack of simple and versatile reverse-genetic methods has limited in vivo functional analysis of candidate genes in this species. Here, we report the establishment of highly efficient and heritable gene mutagenesis methods in the monarch butterfly using transcriptional activator-like effector nucleases (TALENs) and CRISPR-associated RNA-guided nuclease Cas9 (CRISPR/Cas9). Using two clock gene loci, cryptochrome 2 and clock (clk), as candidates, we show that both TALENs and CRISPR/Cas9 generate high-frequency nonhomologous end-joining (NHEJ)-mediated mutations at targeted sites (up to 100%), and that injecting fewer than 100 eggs is sufficient to recover mutant progeny and generate monarch knockout lines in about 3 months. Our study also genetically defines monarch CLK as an essential component of the transcriptional activation complex of the circadian clock. The methods presented should not only greatly accelerate functional analyses of many aspects of monarch biology, but are also anticipated to facilitate the development of these tools in other nontraditional insect species as well as the development of homology-directed knock-ins. PMID:26837953

  19. An insertional mutagenesis system for analyzing the Chinese cabbage genome using Agrobacterium T-DNA.

    PubMed

    Yu, Jae-Gyeong; Lee, Gi-Ho; Kim, Jung-Sun; Shim, Eun-Jo; Park, Young-Doo

    2010-03-01

    In this study, we applied insertional mutagenesis using Agrobacterium transfer DNA to functionally characterize the gene of Brassica rapa L. ssp. pekinensis. The specific objectives were to: (i) develop and apply a gene tagging system using plasmid rescue and inverse PCR, (ii) select and analyze mutant lines, and (iii) analyze the phenotypic characteristics of mutants. A total of 3,400 insertional mutant lines were obtained from the Chinese cabbage cultivar, 'seoul', using optimized condition. Plasmid rescue was performed successfully for transgenic plants with multiple T-DNA insertions, and inverse PCR was performed for plants with a single copy. The isolated flanking DNA sequences were blasted against the NCBI database and mapped to a linkage map. We determined the genetic loci in B. rapa with two methods: RFLP using the rescue clones themselves and sequence homology analysis to the B. rapa sequence database by queries of rescued clones sequences. Compared to wild type, the T(1) progenies of mutant lines showed variable phenotypes, including hairless and wrinkled leaves, rosette-type leaves, and chlorosis symptoms. T-DNA inserted mutant lines were the first population that we developed and will be very useful for functional genomics studies of Chinese cabbage.

  20. Improvement of ENU Mutagenesis Efficiency Using Serial Injection and Mismatch Repair Deficiency Mice.

    PubMed

    Gallego-Llamas, Jabier; Timms, Andrew E; Pitstick, Rose; Peters, Janet; Carlson, George A; Beier, David R

    2016-01-01

    ENU mutagenesis is a powerful method for generating novel lines of mice that are informative with respect to both fundamental biological processes and human disease. Rapid developments in genomic technology have made the task of identifying causal mutations by positional cloning remarkably efficient. One limitation of this approach remains the mutation frequency achievable using standard treatment protocols, which currently generate approximately 1-2 sequence changes per megabase when optimized. In this study we used two strategies to attempt to increase the number of mutations induced by ENU treatment. One approach employed mice carrying a mutation in the DNA repair enzyme Msh6. The second strategy involved injection of ENU to successive generations of mice. To evaluate the number of ENU-induced mutations, single mice or pooled samples were analyzed using whole exome sequencing. The results showed that there is considerable variability in the induced mutation frequency using these approaches, but an overall increase in ENU-induced variants from one generation to another was observed. The analysis of the mice deficient for Msh6 also showed an increase in the ENU-induced variants compared to the wild-type ENU-treated mice. However, in both cases the increase in ENU-induced mutation frequency was modest. PMID:27441645

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. An enhanced system for unnatural amino acid mutagenesis in E. coli.

    PubMed

    Young, Travis S; Ahmad, Insha; Yin, Jun A; Schultz, Peter G

    2010-01-15

    We report a new vector, pEVOL, for the incorporation of unnatural amino acids into proteins in Escherichia coli using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s) (aaRS)/suppressor tRNA pairs. This new system affords higher yields of mutant proteins through the use of both constitutive and inducible promoters to drive the transcription of two copies of the M. jannaschii aaRS gene. Yields were further increased by coupling the dual-aaRS promoter system with a newly optimized suppressor tRNA(CUA)(opt) in a single-vector construct. The optimized suppressor tRNA(CUA)(opt) afforded increased plasmid stability compared with previously reported vectors for unnatural amino acid mutagenesis. To demonstrate the utility of this new system, we introduced 14 mutant aaRS into pEVOL and compared their ability to insert unnatural amino acids in response to three independent amber nonsense codons in sperm whale myoglobin or green fluorescent protein. When cultured in rich media in shake flasks, pEVOL was capable of producing more than 100 mg/L mutant GroEL protein. The versatility, increased yields, and increased stability of the pEVOL vector will further facilitate the expression of proteins with unnatural amino acids. PMID:19852970

  6. A mutagenesis study of the putative luciferin binding site residues of firefly luciferase.

    PubMed

    Branchini, Bruce R; Southworth, Tara L; Murtiashaw, Martha H; Boije, Henrik; Fleet, Sarah E

    2003-09-01

    Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate firefly luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We had previously developed [Branchini, B. R., Magyar, R. A., Murtiashaw, M. H., Anderson, S. M., and Zimmer, M. (1998) Biochemistry 37, 15311-15319] a molecular graphics-based working model of the luciferase active site starting with the first X-ray structure [Conti, E., Franks, N. P., and Brick, P. (1996) Structure 4, 287-298] of the enzyme without bound substrates. In our model, the luciferin binding site contains 15 residues that are within 5 A of the substrate. Using site-directed mutagenesis, we made changes at all of these residues and report here the characterization of the corresponding expressed and purified proteins. Of the 15 residues studied, 12 had a significantly (>or=4-fold K(m) difference) altered binding affinity for luciferin and seven residues, spanning the primary sequence region Arg218-Ala348, had substantially (>or=30 nm) red-shifted bioluminescence emission maxima when mutated. We report here an interpretation of the roles of the mutated residues in substrate binding and bioluminescence color determination. The results of this study generally substantiate the accuracy of our model and provide the foundation for future experiments designed to alter the substrate specificity of firefly luciferase. PMID:12950169

  7. 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

  8. Transposon mutagenesis affecting thiosulfate oxidation in Bosea thiooxidans, a new chemolithoheterotrophic bacterium.

    PubMed

    Das, S K; Mishra, A K

    1996-06-01

    Transposon insertion mutagenesis was used to isolate mutants of Bosea thiooxidans which are impaired in thiosulfate oxidation. Suicide plasmid pSUP5011 was used to introduce the transposon Tn5 into B. thiooxidans via Escherichia coli S17.1-mediated conjugation. Neomycin-resistant transconjugants occurred at a frequency of 2.2 X 10(-4) per donor. Transconjugants defective in thiosulfate oxidation were categorized into three classes on the basis of growth response, enzyme activities, and cytochrome patterns. Class I mutants were deficient in cytochrome c, and no thiosulfate oxidase activity was detected. Class II mutants retained the activities of key enzymes of thiosulfate metabolism, although at reduced levels. Mutants of this class grown on mixed-substrate agar plates deposited elemental sulfur on the colony surfaces. Class III mutants were unable to utilize thiosulfate, though they had normal levels of cytochrome c. The transposon insertions occurred at different chromosomal positions, as confirmed by Southern blotting of chromosomal DNA of mutants deficient in thiosulfate oxidation, a deficiency which resulted from single insertions of Tn5.

  9. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system.

    PubMed

    Liang, Zhen; Zhang, Kang; Chen, Kunling; Gao, Caixia

    2014-02-20

    Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have emerged as powerful tools for genome editing in a variety of species. Here, we report, for the first time, targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, ZmIPK1A, ZmIPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic mutations. Also, we constructed two gRNAs targeting the ZmIPK gene in maize protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition, the CRISPR/Cas system induced targeted mutations in Z. mays protoplasts with efficiencies (13.1%) similar to those obtained with TALENs (9.1%). Our results show that both TALENs and the CRISPR/Cas system can be used for genome modification in maize.

  10. Functional Genomics of Eukaryotic Photosynthesis Using Insertional Mutagenesis of Chlamydomonas reinhardtii1

    PubMed Central

    Dent, Rachel M.; Haglund, Cat M.; Chin, Brian L.; Kobayashi, Marilyn C.; Niyogi, Krishna K.

    2005-01-01

    The unicellular green alga Chlamydomonas reinhardtii is a widely used model organism for studies of oxygenic photosynthesis in eukaryotes. Here we describe the development of a resource for functional genomics of photosynthesis using insertional mutagenesis of the Chlamydomonas nuclear genome. Chlamydomonas cells were transformed with either of two plasmids conferring zeocin resistance, and insertional mutants were selected in the dark on acetate-containing medium to recover light-sensitive and nonphotosynthetic mutants. The population of insertional mutants was subjected to a battery of primary and secondary phenotypic screens to identify photosynthesis-related mutants that were pigment deficient, light sensitive, nonphotosynthetic, or hypersensitive to reactive oxygen species. Approximately 9% of the insertional mutants exhibited 1 or more of these phenotypes. Molecular analysis showed that each mutant line contains an average of 1.4 insertions, and genetic analysis indicated that approximately 50% of the mutations are tagged by the transforming DNA. Flanking DNA was isolated from the mutants, and sequence data for the insertion sites in 50 mutants are presented and discussed. PMID:15653810

  11. Probing the chemical mechanism of saccharopine reductase from Saccharomyces cerevisiae using site-directed mutagenesis.

    PubMed

    Vashishtha, Ashwani K; West, Ann H; Cook, Paul F

    2015-10-15

    Saccharopine reductase catalyzes the reductive amination of l-α-aminoadipate-δ-semialdehyde with l-glutamate to give saccharopine. Two mechanisms have been proposed for the reductase, one that makes use of enzyme side chains as acid-base catalytic groups, and a second, in which the reaction is catalyzed by enzyme-bound reactants. Site-directed mutagenesis was used to change acid-base candidates in the active site of the reductase to eliminate their ionizable side chain. Thus, the D126A, C154S and Y99F and several double mutant enzymes were prepared. Kinetic parameters in the direction of glutamate formation exhibited modest decreases, inconsistent with the loss of an acid-base catalyst. The pH-rate profiles obtained with all mutant enzymes decrease at low and high pH, suggesting acid and base catalytic groups are still present in all enzymes. Solvent kinetic deuterium isotope effects are all larger than those observed for wild type enzyme, and approximately equal to one another, suggesting the slow step is the same as that of wild type enzyme, a conformational change to open the site and release products (in the direction of saccharopine formation). Overall, the acid-base chemistry is likely catalyzed by bound reactants, with the exception of deprotonation of the α-amine of glutamate, which likely requires an enzyme residue. PMID:26342457