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Sample records for modeling site-directed mutagenesis

  1. How batrachotoxin modifies the sodium channel permeation pathway: computer modeling and site-directed mutagenesis.

    Science.gov (United States)

    Wang, Sho-Ya; Mitchell, Jane; Tikhonov, Denis B; Zhorov, Boris S; Wang, Ging Kuo

    2006-03-01

    A structural model of the rNav1.4 Na+ channel with batrachotoxin (BTX) bound within the inner cavity suggested that the BTX pyrrole moiety is located between a lysine residue at the DEKA selectivity filter (Lys1237) and an adjacent phenylalanine residue (Phe1236). We tested this pyrrole-binding model by site-directed mutagenesis of Phe1236 at D3/P-loop with 11 amino acids. Mutants F1236D and F1236E expressed poorly, whereas nine other mutants either expressed robust Na+ currents, like the wild-type (F1236Y/Q/K), or somewhat reduced current (F1236G/A/C/N/W/R). Gating properties were altered modestly in most mutant channels, with F1236G displaying the greatest shift in activation and steady-state fast inactivation (-10.1 and -7.5 mV, respectively). Mutants F1236K and F1236R were severely resistant to BTX after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz), whereas seven other mutants were sensitive but with reduced magnitudes compared with the wild type. It is noteworthy that rNav1.4-F1236K mutant Na+ channels remained highly sensitive to block by the local anesthetic bupivacaine, unlike several other BTX-resistant mutant channels. Our data thus support a model in which BTX, when bound within the inner cavity, interacts with the D3/P-loop directly. Such a direct interaction provides clues on how BTX alters the Na+ channel selectivity and conductance.

  2. Efficient multi-site-directed mutagenesis directly from genomic ...

    Indian Academy of Sciences (India)

    2012-09-29

    Sep 29, 2012 ... overlap extension PCR (OE-PCR) and Quick-change multi- site-directed mutagenesis systems developed by Stratagene. Company are predominately used owing to their simplicity and efficiency. Quick-change method is simple for MSM, but it requires circular plasmid as an amplification template.

  3. The active site and substrate-binding mode of 1-aminocyclopropane-1-carboxylate oxidase determined by site-directed mutagenesis and comparative modelling studies.

    Science.gov (United States)

    Seo, Young Sam; Yoo, Ahrim; Jung, Jinwon; Sung, Soon-Kee; Yang, Dae Ryook; Kim, Woo Taek; Lee, Weontae

    2004-01-01

    The active site and substrate-binding mode of MD-ACO1 (Malus domestica Borkh. 1-aminocyclopropane-1-carboxylate oxidase) have been determined using site-directed mutagenesis and comparative modelling methods. The MD-ACO1 protein folds into a compact jelly-roll motif comprised of eight a-helices, 12 b-strands and several long loops. The active site is well defined as a wide cleft near the C-terminus. The co-substrate ascorbate is located in cofactor Fe2+-binding pocket, the so-called '2-His-1-carboxylate facial triad'. In addition, our results reveal that Arg244 and Ser246 are involved in generating the reaction product during enzyme catalysis. The structure agrees well with the biochemical and site-directed mutagenesis results. The three-dimensional structure together with the steady-state kinetics of both the wild-type and mutant MD-ACO1 proteins reveal how the substrate specificity of MD-ACO1 is involved in the catalytic mechanism, providing insights into understanding the fruit ripening process at atomic resolution. PMID:14972027

  4. Site-directed mutagenesis and molecular modelling studies show the role of Asp82 and cysteines in rat acylase 1, a member of the M20 family

    International Nuclear Information System (INIS)

    Herga, Sameh; Brutus, Alexandre; Vitale, Rosa Maria; Miche, Helene; Perrier, Josette; Puigserver, Antoine; Scaloni, Andrea; Giardina, Thierry

    2005-01-01

    Acylase 1 from rat kidney catalyzes the hydrolysis of acyl-amino acids. Sequence alignment has shown that this enzyme belongs to the metalloprotein family M20. Site-directed mutagenesis experiments led to the identification of one functionally important amino acid residue located near one of the zinc coordinating residues, which play a critical role in the enzymatic activity. The D82N- and D82E-substituted forms showed no significant activity and very low activity, respectively, along with a loss of zinc coordination. Molecular modelling investigations indicated a putative role of D82 in ensuring a proper protonation of catalytic histidine. In addition, none of the five cysteine residues present in the rat kidney acylase 1 sequence seemed involved in the catalytic process: the loss of activity induced by the C294A substitution was probably due to a conformational change in the 3D structure

  5. The peptide agonist-binding site of the glucagon-like peptide-1 (GLP-1) receptor based on site-directed mutagenesis and knowledge-based modelling.

    Science.gov (United States)

    Dods, Rachel L; Donnelly, Dan

    2015-11-23

    Glucagon-like peptide-1 (7-36)amide (GLP-1) plays a central role in regulating blood sugar levels and its receptor, GLP-1R, is a target for anti-diabetic agents such as the peptide agonist drugs exenatide and liraglutide. In order to understand the molecular nature of the peptide-receptor interaction, we used site-directed mutagenesis and pharmacological profiling to highlight nine sites as being important for peptide agonist binding and/or activation. Using a knowledge-based approach, we constructed a 3D model of agonist-bound GLP-1R, basing the conformation of the N-terminal region on that of the receptor-bound NMR structure of the related peptide pituitary adenylate cyclase-activating protein (PACAP21). The relative position of the extracellular to the transmembrane (TM) domain, as well as the molecular details of the agonist-binding site itself, were found to be different from the model that was published alongside the crystal structure of the TM domain of the glucagon receptor, but were nevertheless more compatible with published mutagenesis data. Furthermore, the NMR-determined structure of a high-potency cyclic conformationally-constrained 11-residue analogue of GLP-1 was also docked into the receptor-binding site. Despite having a different main chain conformation to that seen in the PACAP21 structure, four conserved residues (equivalent to His-7, Glu-9, Ser-14 and Asp-15 in GLP-1) could be structurally aligned and made similar interactions with the receptor as their equivalents in the GLP-1-docked model, suggesting the basis of a pharmacophore for GLP-1R peptide agonists. In this way, the model not only explains current mutagenesis and molecular pharmacological data but also provides a basis for further experimental design. © 2016 Authors.

  6. Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesis.

    Science.gov (United States)

    Mattern-Dogru, Emine; Ma, Xueyan; Hartmann, Joachim; Decker, Heinz; Stöckigt, Joachim

    2002-06-01

    In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the alpha/beta hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By site-directed mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the alpha/beta hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the alpha/beta hydrolase enzyme superfamily.

  7. Site-directed mutagenesis under the direction of in silico protein docking modeling reveals the active site residues of 3-ketosteroid-Δ1-dehydrogenase from Mycobacterium neoaurum.

    Science.gov (United States)

    Qin, Ning; Shen, Yanbing; Yang, Xu; Su, Liqiu; Tang, Rui; Li, Wei; Wang, Min

    2017-07-01

    3-Ketosteroid-Δ 1 -dehydrogenases (KsdD) from Mycobacterium neoaurum could transform androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione. This reaction has a significant effect on the product of pharmaceutical steroid. The crystal structure and active site residues information of KsdD from Mycobacterium is not yet available, which result in the engineering of KsdD is tedious. In this study, by the way of protein modeling and site-directed mutagenesis, we find that, Y122, Y125, S138, E140 and Y541 from the FAD-binding domain and Y365 from the catalytic domain play a key role in this transformation. Compared with the wild type, the decline in AD conversion for mutants illustrated that Y125, Y365, and Y541 were essential to the function of KsdD. Y122, S138 and E140 contributed to the catalysis of KsdD. The following analysis revealed the catalysis mechanism of these mutations in KsdD of Mycobacterium. These information presented here facilitate the manipulation of the catalytic properties of the enzyme to improve its application in the pharmaceutical steroid industry.

  8. Construction of a high-efficiency multi-site-directed mutagenesis ...

    African Journals Online (AJOL)

    Although site-directed mutagenesis has been used in many fields, it still has low rate of success and high cost because of low-yield target products. A modified method for multi-site-directed mutagenesis was developed with shifted primer design and cold-start polymerase chain reaction (PCR). The developed method was ...

  9. Expression and site-directed mutagenesis of human dihydrofolate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Prendergast, N.J.; Delcamp, T.J.; Smith, P.L.; Freisheim, J.H.

    1988-05-17

    A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 ..-->.. Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by ..cap alpha..-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.

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

    Directory of Open Access Journals (Sweden)

    Janire Mingo

    Full Text Available 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.

  11. Software-Supported USER Cloning Strategies for Site-Directed Mutagenesis and DNA Assembly

    DEFF Research Database (Denmark)

    Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen

    2015-01-01

    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 ....

  12. An In Vitro Single-Primer Site-Directed Mutagenesis Method for Use in Biotechnology.

    Science.gov (United States)

    Huang, Yanchao; Zhang, Likui

    2017-01-01

    Site-directed mutagenesis is a powerful method to introduce mutation(s) into DNA sequences. A number of methods have been developed over the years with a main goal being to create a high number of mutant genes. The single-mutagenic primer method for site-directed mutagenesis is the most direct method that yields mutant genes in about 25-50 % of transformants in a robust, low-cost reaction. The supercompetent XL10-Gold bacteria used in the Stratagene protocol carry a phage, which may be a problem for some applications; however, in our single-mutagenic primer method the supercompetent bacteria are not needed. A thermostable DNA polymerase with high fidelity and processivity, such as Phusion DNA polymerase, is required for our optimized procedure to avoid extra mutation(s) and enhance mutagenic efficiency.

  13. Mechanism of adenylate kinase: Site-directed mutagenesis versus x-ray and NMR

    International Nuclear Information System (INIS)

    Tsai, Mingdaw; Yan, Honggao

    1991-01-01

    Controversy is an integral part of scientific research and is often a precursor to the truth. However, this lesson has been learned in a very hard way in the case of the structure-function relationship of adenylate kinase (AK), which catalyzes the interconversion between MgATP+AMP and MgADP+ADP. While this small kinase has been considered a model kinase and the enzyme-substrate interaction of AK was among the first investigated by X-ray crystallography and NMR the substrate binding sites deduced from the early studies by these two powerful techniques (termed the X-ray model and the NMR model, respectively) were dramatically different. Ironically, both models have had substantial impact on researchers in related fields. The problems have finally been dealt with since 1987 by the interplay between site-directed mutagenesis, X-ray, and NMR. The purpose of this review is not only to summarize the current knowledge in the structure-function relationship of adenylate kinase but also to accurately document and critically analyze historical developments in the hope that history will not be repeated

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Efficient method for site-directed mutagenesis in large plasmids without subcloning.

    Directory of Open Access Journals (Sweden)

    Louay K Hallak

    Full Text Available Commonly used methods for site-directed DNA mutagenesis require copying the entire target plasmid. These methods allow relatively easy modification of DNA sequences in small plasmids but become less efficient and faithful for large plasmids, necessitating full sequence verification. Introduction of mutations in larger plasmids requires subcloning, a slow and labor-intensive process, especially for multiple mutations. We have developed an efficient DNA mutagenesis technique, UnRestricted Mutagenesis and Cloning (URMAC that replaces subcloning steps with quick biochemical reactions. URMAC does not suffer from plasmid size constraints and allows simultaneous introduction of multiple mutations. URMAC involves manipulation of only the mutagenesis target site(s, not the entire plasmid being mutagenized, therefore only partial sequence verification is required. Basic URMAC requires two PCR reactions, each followed by a ligation reaction to circularize the product, with an optional third enrichment PCR step followed by a traditional cloning step that requires two restriction sites. Here, we demonstrate URMAC's speed, accuracy, and efficiency through several examples, creating insertions, deletions or substitutions in plasmids ranging from 2.6 kb to 17 kb without subcloning.

  16. Efficient method for site-directed mutagenesis in large plasmids without subcloning.

    Science.gov (United States)

    Hallak, Louay K; Berger, Kelly; Kaspar, Rita; Kwilas, Anna R; Montanaro, Federica; Peeples, Mark E

    2017-01-01

    Commonly used methods for site-directed DNA mutagenesis require copying the entire target plasmid. These methods allow relatively easy modification of DNA sequences in small plasmids but become less efficient and faithful for large plasmids, necessitating full sequence verification. Introduction of mutations in larger plasmids requires subcloning, a slow and labor-intensive process, especially for multiple mutations. We have developed an efficient DNA mutagenesis technique, UnRestricted Mutagenesis and Cloning (URMAC) that replaces subcloning steps with quick biochemical reactions. URMAC does not suffer from plasmid size constraints and allows simultaneous introduction of multiple mutations. URMAC involves manipulation of only the mutagenesis target site(s), not the entire plasmid being mutagenized, therefore only partial sequence verification is required. Basic URMAC requires two PCR reactions, each followed by a ligation reaction to circularize the product, with an optional third enrichment PCR step followed by a traditional cloning step that requires two restriction sites. Here, we demonstrate URMAC's speed, accuracy, and efficiency through several examples, creating insertions, deletions or substitutions in plasmids ranging from 2.6 kb to 17 kb without subcloning.

  17. Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis.

    Science.gov (United States)

    Mohammadi, Mohsen; Sepehrizadeh, Zargham; Ebrahim-Habibi, Azadeh; Shahverdi, Ahmad Reza; Faramarzi, Mohammad Ali; Setayesh, Neda

    2016-11-01

    Lipases as significant biocatalysts had been widely employed to catalyze various chemical reactions such as ester hydrolysis, ester synthesis, and transesterification. Improving the activity and thermostability of enzymes is desirable for industrial applications. The lipase of Serratia marcescens belonging to family I.3 lipase has a very important pharmaceutical application in production of chiral precursors. In the present study, to achieve improved lipase activity and thermostability, using computational predictions of protein, four mutant lipases of SML (MutG2P, MutG59P, Mut H279K and MutL613WA614P) were constructed by site-directed mutagenesis. The recombinant mutant proteins were over-expressed in E. coli and purified by affinity chromatography on the Ni-NTA system. Circular dichroism spectroscopy, differential scanning calorimetry and kinetic parameters (Km and kcat) were determined. Our results have shown that the secondary structure of all lipases was approximately similar to one another. The MutG2P and MutG59P were more stable than wild type by approximately 2.3 and 2.9 in T 1/2 , respectively. The catalytic efficiency (kcat/Km) of MutH279K was enhanced by 2-fold as compared with the wild type (p<0.05). These results indicate that using protein modeling program and creating mutation, can enhance lipase activity and/or thermostability of SML and it also could be used for improving other properties of enzyme to the desired requirements as well as further mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Crucial role of the orexin-B C-terminus in the induction of OX1 receptor-mediated apoptosis: analysis by alanine scanning, molecular modelling and site-directed mutagenesis.

    Science.gov (United States)

    Nicole, Pascal; Couvineau, Pierre; Jamin, Nadege; Voisin, Thierry; Couvineau, Alain

    2015-11-01

    Orexins (A and B) are hypothalamic peptides that interact with OX1 and OX2 receptors and are involved in the sleep/wake cycle. We previously demonstrated that OX1 receptors are highly expressed in colon cancer tumours and colonic cancer cell lines where orexins induce apoptosis and inhibit tumour growth in preclinical animal models. The present study explored the structure-function relationships of orexin-B and OX1 receptors. The contribution of all orexin-B residues in orexin-B-induced apoptosis was investigated by alanine scanning. To determine which OX1 receptor domains are involved in orexin-B binding and apoptosis, a 3D model of OX1 receptor docked to the orexin-B C-terminus (AA-20-28) was developed. Substitution of residues present in OX1 receptor transmembrane (TM) domains by site-directed mutagenesis was performed. Alanine substitution of orexin-B residues, L(11) , L(15) , A(22) , G(24) , I(25) , L(26) and M(28) , altered orexin-B's binding affinity. Substitution of these residues and of the Q(16) , A(17) , S(18) , N(20) and T(27) residues inhibited apoptosis in CHO-S-OX1 receptor cells. The K(120) , P(123) , Y(124) , N(318) , K(321) , F(340) , T(341) , H(344) and W(345) residues localized in TM2, TM3, TM6 and TM7 of OX1 receptors were shown to play a role in orexin-B recognition and orexin-B/OX1 receptor-induced apoptosis. The C-terminus of orexin-B (i) plays an important role in its pro-apoptotic effect; and (ii) interacts with some residues localized in the OX1 receptor TM. This study defines the structure-function relationship for orexin-B recognition by human OX1 receptors and orexin-B/OX1 receptor-induced apoptosis, an important step for the future development of new agonist molecules. © 2015 The British Pharmacological Society.

  19. Color transitions in coral's fluorescent proteins by site-directed mutagenesis

    Directory of Open Access Journals (Sweden)

    Lukyanov Sergey A

    2001-07-01

    Full Text Available Abstract Background Green Fluorescent Protein (GFP cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (~ 485 nm, green (~ 505 nm, yellow (~ 540 nm, and red (>580 nm emitters. Results Here we applied site-directed mutagenesis in order to investigate the structural background of color variety and possibility of shifting between different types of fluorescence. First, a blue-shifted mutant of cyan amFP486 was generated. Second, it was established that cyan and green emitters can be modified so as to produce an intermediate spectrum of fluorescence. Third, the relationship between green and yellow fluorescence was inspected on closely homologous green zFP506 and yellow zFP538 proteins. The following transitions of colors were performed: yellow to green; yellow to dual color (green and yellow; and green to yellow. Fourth, we generated a mutant of cyan emitter dsFP483 that demonstrated dual color (cyan and red fluorescence. Conclusions Several amino acid substitutions were found to strongly affect fluorescence maxima. Some positions primarily found by sequence comparison were proved to be crucial for fluorescence of particular color. These results are the first step towards predicting the color of natural GFP-like proteins corresponding to newly identified cDNAs from corals.

  20. SDM-Assist software to design site-directed mutagenesis primers introducing "silent" restriction sites.

    Science.gov (United States)

    Karnik, Abhijit; Karnik, Rucha; Grefen, Christopher

    2013-03-22

    Over the past decades site-directed mutagenesis (SDM) has become an indispensable tool for biological structure-function studies. In principle, SDM uses modified primer pairs in a PCR reaction to introduce a mutation in a cDNA insert. DpnI digestion of the reaction mixture is used to eliminate template copies before amplification in E. coli; however, this process is inefficient resulting in un-mutated clones which can only be distinguished from mutant clones by sequencing. We have developed a program - 'SDM-Assist' which creates SDM primers adding a specific identifier: through additional silent mutations a restriction site is included or a previous one removed which allows for highly efficient identification of 'mutated clones' by a simple restriction digest. The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the primers based on factors such as Tm, GC content and secondary structure allowing for simplified selection of optimal primer pairs.

  1. Tailor-Made Protein Tyrosine Phosphatases: In Vitro Site-Directed Mutagenesis of PTEN and PTPRZ-B

    NARCIS (Netherlands)

    Luna, S.; Mingo, J.; Aurtenetxe, O.; Blanco, L.; Amo, L.; Schepens, J.; Hendriks, W.J.A.J.; Pulido, R.

    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

  2. New insights into the QuikChange™ process guide the use of Phusion DNA polymerase for site-directed mutagenesis.

    Science.gov (United States)

    Xia, Yongzhen; Chu, Wenqiao; Qi, Qingsheng; Xun, Luying

    2015-01-01

    The QuikChange™ site-directed mutagenesis method is popular but imperfect. An improvement by using partially overlapping primers has been reported several times; however, it is incompatible with the proposed mechanism. The QuikChange™ method using complementary primers is proposed to linearly amplify a target plasmid with the products annealing to produce double-stranded DNA molecules with 5'-overhangs. The overhang annealing is supposed to form circular plasmids with staggered breaks, which can be repaired in Escherichia coli after transformation. Here, we demonstrated that the PCR enzyme fills the 5'-overhangs in the early cycles, and the product is then used as the template for exponential amplification. The linear DNA molecules with homologous ends are joined to generate the plasmid with the desired mutations through homologous recombination in E. coli. The correct understanding is important to method improvements, guiding us to use partially overlapping primers and Phusion DNA polymerase for site-directed mutagenesis. Phusion did not amplify a plasmid with complementary primers but used partially overlapping primers to amplify the plasmid, producing linear DNA molecules with homologous ends for site-directed mutagenesis. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. HTP-OligoDesigner: An Online Primer Design Tool for High-Throughput Gene Cloning and Site-Directed Mutagenesis.

    Science.gov (United States)

    Camilo, Cesar M; Lima, Gustavo M A; Maluf, Fernando V; Guido, Rafael V C; Polikarpov, Igor

    2016-01-01

    Following burgeoning genomic and transcriptomic sequencing data, biochemical and molecular biology groups worldwide are implementing high-throughput cloning and mutagenesis facilities in order to obtain a large number of soluble proteins for structural and functional characterization. Since manual primer design can be a time-consuming and error-generating step, particularly when working with hundreds of targets, the automation of primer design process becomes highly desirable. HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive online primer design tool for both laboratory-scale and high-throughput projects of sequence-independent gene cloning and site-directed mutagenesis and a Tm calculator for quick queries.

  4. Increase in D-tagatose production rate by site-directed mutagenesis of L-arabinose isomerase from Geobacillus thermodenitrificans.

    Science.gov (United States)

    Oh, Hyo-Jung; Kim, Hye-Jung; Oh, Deok-Kun

    2006-02-01

    Among single-site mutations of L-arabinose isomerase derived from Geobacillus thermodenitrificans, two mutants were produced having the lowest and highest activities of D-tagatose production. Site-directed mutagenesis at these sites showed that the aromatic ring at amino acid 164 and the size of amino acid 475 were important for D-tagatose production. Among double-site mutations, one mutant converted D-galactose into D-tagatose with a yield of 58% whereas the wild type gave 46% D-tagatose conversion after 300 min at 65 degrees C.

  5. Protein crystallography and site-direct mutagenesis analysis of the poly(ethylene terephthalate) hydrolase PETase from Ideonella sakaiensis.

    Science.gov (United States)

    Liu, Bing; He, Lihui; Wang, Liping; Li, Tao; Li, Changcheng; Liu, Huayi; Luo, Yunzi; Bao, Rui

    2018-03-30

    Compared with traditional recycle strategies, biodegradation provides a sustainable solution for poly (ethylene terephthalate) (PET) wastes disposal. PETase, a newly identified enzyme from Ideonella sakaiensis, has high efficiency and specificity towards PET, which provides a prominent prospect on PET degradation. Based on the biochemical analysis, we propose that the wide substrate-binding pocket is critical for its excellent property on crystallized PET hydrolysis. Structure-guided site-directed mutagenesis exhibited improvement in PETase catalytic efficiency, providing valuable insight on how the molecular engineering of PETase can optimize its application in biocatalysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Site-directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of Rab7b paralogue in the model eukaryote Paramecium octaurelia.

    Science.gov (United States)

    Wyroba, E; Kwaśniak, P; Miller, K; Kobyłecki, K; Osińska, M

    2016-04-11

    Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue - distinct from that of Rab7a directly involved in phagocytosis - was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala200 resulted in diminished incorporation of [P32] by 37.4% and of 32 [C14-]UDP-glucose by 24%, respectively, into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells contrary to non- mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.63+ characteristic for the glycan group attached to Thr200 in Rab7b using nano LC-MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P32]and  [C14-]UDP- glucose, the suggested composition of the adduct attached to Thr200 might be (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Rab7b in Paramecium is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a.

  7. Specificity of N-terminal methionyl peptidase: analysis by site-directed mutagenesis

    International Nuclear Information System (INIS)

    Kasper, T.J.; Boissel, J.P.; Bunn, H.F.

    1987-01-01

    The start site of eukaryotic translation is normally an AUG codon. The corresponding N-terminal methionine is most often removed when the nascent chain reaches about 30 residues. Data from a survey of 1764 eukaryotic protein sequences suggest that the residue adjacent to the initiator Met determines Met cleavage. In order to investigate the mechanism of this reaction, the authors have prepared oligonucleotide-directed mutants of human β-globin from gapped heteroduplexes of a T3/T7 plasmid containing a globin cDNA clone. To date, the authors have produced mutants encoding for 15 of 19 possible amino acid replacements at position 1 in the β-globin chain. These mutants have been confirmed by dideoxy sequencing, transcribed in vitro, and translated in a rabbit reticulocyte lysate in the presence of 35 S-methionine. Labeled translation products were then isolated by cation exchange HPLC, and tryptic peptides were analyzed by RP-HPLC. Thus far, this structural analysis has shown that for β-1 Val, Ala, and Ser, the initiator Met is cleaved, whereas for β-1 Lys, Met, Glu, Trp, Asn, Tyr, and Glu, initiator Met is retained. For β-1 Leu initiator Met is cleaved with a frequency of about 50%. These results are consistent with the data obtained from the previous survey. The expression of site-directed mutants in a cell-free system can also be used to investigate other N-terminal processing events, such as acetylation and myristylation

  8. Changing the inhibitory specificity and function of Cucurbita maxima trypsin inhibitor-V by site-directed mutagenesis.

    Science.gov (United States)

    Wen, L; Lee, I; Chen, G; Huang, J K; Gong, Y; Krishnamoorthi, R

    1995-02-27

    Cucurbita maxima trypsin inhibitor-V (CMTI-V) is also a specific inhibitor of human blood coagulation factor beta-factor XIIa. A recombinant version of CMTI-V has allowed probing of roles of individual amino acid residues including the reactive site residue, lysine (P1), by site-directed mutagenesis. The K44R showed at least a 5-fold increase in inhibitory activity toward human beta-factor XIIa, while there was no change toward bovine trypsin. This result demonstrates that beta-factor-XIIa prefers an arginine residue over lysine residue, while trypsin is non-specific to lysine or arginine in its binding pocket. On the other hand, the specificity of CMTI-V could be changed from trypsin to chymotrypsin inhibition by mutation of the P1 residue to either leucine or methionine (K44L or K44M).

  9. Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

    OpenAIRE

    片岡, 邦重; Kataoka, Kunishige; Tanizawa, Katsuyuki

    2003-01-01

    The residues L40, A113, V291, and V294, in leucine dehydrogenase (LeuDH), predicted to be involved in recognition of the substrate side chain, have been mutated on the basis of the molecular modeling to mimic the substrate specificities of phenylalanine (PheDH), glutamate (GluDH), and lysine dehydrogenases (LysDH). The A113G and A113G/V291L mutants, imitating the PheDH active site, displayed activities toward -phenylalanine and phenylpyruvate with 1.6 and 7.8% of kcat values of the wild-type ...

  10. MALS: an efficient strategy for multiple site-directed mutagenesis employing a combination of DNA amplification, ligation and suppression PCR

    Directory of Open Access Journals (Sweden)

    Drayna Dennis T

    2009-09-01

    Full Text Available Abstract Background Multiple approaches for the site-directed mutagenesis (SDM have been developed. However, only several of them are designed for simultaneous introduction of multiple nucleotide alterations, and these are time consuming. In addition, many of the existing multiple SDM methods have technical limitations associated with type and number of mutations that can be introduced, or are technically demanding and require special chemical reagents. Results In this study we developed a quick and efficient strategy for introduction of multiple complex mutations in a target DNA without intermediate subcloning by using a combination of connecting SDM and suppression PCR. The procedure consists of sequential rounds, with each individual round including PCR amplification of target DNA with two non-overlapping pairs of oligonucleotides. The desired mutation is incorporated at the 5' end of one or both internal oligonucleotides. DNA fragments obtained during amplification are mixed and ligated. The resulting DNA mixture is amplified with external oligonucleotides that act as suppression adapters. Suppression PCR limits amplification to DNA molecules representing full length target DNA, while amplification of other types of molecules formed during ligation is suppressed. To create additional mutations, an aliquot of the ligation mixture is then used directly for the next round of mutagenesis employing internal oligonucleotides specific for another region of target DNA. Conclusion A wide variety of complex multiple mutations can be generated in a short period of time. The procedure is rapid, highly efficient and does not require special chemical reagents. Thus, MALS represents a powerful alternative to the existing methods for multiple SDM.

  11. Characterization of vanadium-binding sites of the vanadium-binding protein Vanabin2 by site-directed mutagenesis.

    Science.gov (United States)

    Ueki, Tatsuya; Kawakami, Norifumi; Toshishige, Masaaki; Matsuo, Koichi; Gekko, Kunihiko; Michibata, Hitoshi

    2009-10-01

    Vanabins are a unique protein family of vanadium-binding proteins with nine disulfide bonds. Possible binding sites for VO2+ in Vanabin2 from a vanadium-rich ascidian Ascidia sydneiensis samea have been detected by nuclear magnetic resonance study, but the metal selectivity and metal-binding ability of each site was not examined. In order to reveal functional contribution of each binding site, we prepared several mutants of Vanabin2 by in vitro site-directed mutagenesis and analyzed their metal selectivity and affinity by immobilized metal-ion affinity chromatography and Hummel Dreyer method. Mutation at K10/R60 (site 1) markedly reduced the affinity for VO2+. Mutation at K24/K38/R41/R42 (site 2) decreased the maximum binding number, but only slightly increased the overall affinity for VO2+. Secondary structure of both mutants was the same as that of the wild type as assessed by circular dichroism spectroscopy. Mutation in disulfide bonds near the site 1 did not affect its high affinity binding capacity, while those near the site 2 decreased the overall affinity for VO2+. These results suggested that the site 1 is a high affinity binding site for VO2+, while the site 2 composes a moderate affinity site for multiple VO2+.

  12. Modifying the photoelectric behavior of bacteriorhodopsin by site-directed mutagenesis: electrochemical and genetic engineering approaches to molecular devices

    Science.gov (United States)

    Hong, F. T.; Hong, F. H.; Needleman, R. B.; Ni, B.; Chang, M.

    1992-07-01

    Bacteriorhodopsins (bR's) modified by substitution of the chromophore with synthetic vitamin A analogues or by spontaneous mutation have been reported as successful examples of using biomaterials to construct molecular optoelectronic devices. The operation of these devices depends on desirable optical properties derived from molecular engineering. This report examines the effect of site-directed mutagenesis on the photoelectric behavior of bR thin films with an emphasis on their application to the construction of molecular devices based on their unique photoelectric behavior. We examine the photoelectric signals induced by a microsecond light pulse in thin films which contain reconstituted oriented purple membrane sheets isolated from several mutant strains of Halobacterium halobium. A recently developed expression system is used to synthesize mutant bR's in their natural host, H. halobium. We then use a unique analytical method (tunable voltage clamp method) to investigate the effect of pH on the relaxation of two components of the photoelectric signals, B1 and B2. We found that for the four mutant bR's examined, the pH dependence of the B2 component varies significantly. Our results suggest that genetic engineering approaches can produce mutant bR's with altered photoelectric characteristics that can be exploited in the construction of devices.

  13. Site-directed Mutagenesis of Cysteine Residues in Phi-class Glutathione S-transferase F3 from Oryza sativa

    Energy Technology Data Exchange (ETDEWEB)

    Jo, Hyunjoo; Lee, Juwon; Noh, Jinseok; Kong, Kwanghoon [Chung-Ang Univ., Seoul (Korea, Republic of)

    2012-12-15

    To elucidate the roles of cysteine residues in rice Phi-class GST F3, in this study, all three cysteine residues were replaced with alanine by site-directed mutagenesis in order to obtain mutants C22A, C73A and C77A. Three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitutions of Cys73 and Cys77 residues in OsGSTF3 with alanine did not affect the glutathione conjugation activities, showing non-essentiality of these residues. On the other hand, the substitution of Cys22 residue with alanine resulted in approximately a 60% loss of specific activity toward ethacrynic acid. Moreover, the K{sub m}{sup CDNB} value of the mutant C22A was approximately 2.2 fold larger than that of the wild type. From these results, the evolutionally conserved cysteine 22 residue seems to participate rather in the structural stability of the active site in OsGSTF3 by stabilizing the electrophilic substrates-binding site's conformation than in the substrate binding directly.

  14. Assignment of histidine resonances in the 1H NMR (500 MHz) spectrum of subtilisin BPN' using site-directed mutagenesis

    International Nuclear Information System (INIS)

    Bycroft, M.; Fersht, A.R.

    1988-01-01

    A spin-echo pulse sequence has been used to resolve the six histidine C-2H protons in the 500-MHz NMR spectrum of subtilisin BPN'. Five of these residues have been substituted by site-directed mutagenesis, and this has enabled a complete assignment of these protons to be obtained. Analysis of the pH titration curves of these signals has provided microscopic pK a 's for the six histidines in this enzyme. The pK a 's of the histidine residues in subtilisin BPN' have been compared with the values obtained for the histidines in the homologous enzyme from Bacillus licheniformis (subtilisin Carlsberg). Four of the five conserved histidines titrate with essentially identical pK a 's in the two enzymes. It therefore appears that the assignments made for these residues in subtilisin BPN' can be transferred to subtilisin Carlsberg. On the basis of these assignments, the one histidine that titrates with a substantially different pK a in the two enzymes can be assigned to histidine-238. This difference in pK a has been attributed to a Trp to Lys substitution at position 241 in subtilisin Carlsberg

  15. Site directed mutagenesis of amino acid residues at the active site of mouse aldehyde oxidase AOX1.

    Directory of Open Access Journals (Sweden)

    Silvia Schumann

    Full Text Available Mouse aldehyde oxidase (mAOX1 forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Escherichia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.

  16. Improving the Catalytic Behavior of DFA I-Forming Inulin Fructotransferase from Streptomyces davawensis with Site-Directed Mutagenesis.

    Science.gov (United States)

    Yu, Shuhuai; Zhang, Yanmin; Zhu, Yingying; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2017-08-30

    Previously, a α-d-fructofuranose-β-d-fructofuranose 1,2':2,1'-dianhydride (DFA I)-forming inulin fructotransferase (IFTase), namely, SdIFTase, was identified. The enzyme does not show high performances. In this work, to improve catalytic behavior including activity and thermostability, the enzyme was modified using site-directed mutagenesis on the basis of structure. The mutated residues were divided into three groups. Those in group I are located at central tunnel including G236, A257, G281, T313, and A314S. The group II contains residues at the inner edge of substrate binding pocket including I80, while group III at the outer edge includes G121 and T122. The thermostability was reflected by the melting temperature (T m ) determined by Nano DSC. Finally, the T m values of G236S/G281S/A257S/T313S/A314S in group I and G121A/T122L in group III were enhanced by 3.2 and 4.5 °C, and the relative activities were enhanced to 140.5% and 148.7%, respectively. The method in this work may be applicable to other DFA I-forming IFTases.

  17. Characterization of recombinant human α2-antiplasmin and of mutants obtained by site-directed mutagenesis of the reactive site

    International Nuclear Information System (INIS)

    Holmes, W.E.; Lijnen, H.R.; Collen, D.

    1987-01-01

    Human α 2 -antiplasmin (α 2 AP) has been expressed in Chinese hamster ovary cells and purified from conditioned media. The recombinant protein (rα 2 AP) is immunologically identical with natural α 2 AP and indistinguishable with respect to plasmin(ogen) binding properties. Second-order rate constants (k 1 ) for the interaction of α 2 AP and rα 2 AP with plasmin are both (1-2) x 10 7 M -1 s -1 . In order to examine the effects of alterations within the reactive site of α 2 AP, deletions of the P 1 residue Arg-364 (rα 2 AP-ΔArg364) of the P' 1 residue Met-365 (rα 2 AP-ΔMet365) were introduced by in vitro site-directed mutagenesis. rα 2 AP-ΔMet365 completely retains its ability to inhibit both plasmin and trypsin, indicating that α 2 AP has no absolute requirement for Met in the P' 1 position. Unexpectedly, no increase in antithrombin activity was observed. rα 2 AP-ΔArg364 has lost the ability to inhibit plasmin, trypsin, and thrombin, but unlike the wild-type protein, this variant is an effective elastase inhibitor

  18. Site-directed mutagenesis of the histamine H1-receptor reveals a selective interaction of asparagine207 with subclasses of H1-receptor agonists

    NARCIS (Netherlands)

    Leurs, R; Smit, M J; Tensen, C P; Ter Laak, A M; Timmerman, H

    1994-01-01

    In this study we investigated the role of the threonine203 and the asparagine207 residues in the fifth transmembrane domain of the guinea-pig histamine H1-receptor by site-directed mutagenesis to non-functional alanines. Whereas the threonine203 residue is not important for the action of histamine,

  19. Site-Directed Mutagenesis Analysis of Amino Acids Critical for Activity of the Type I Signal Peptidase of the Archaeon Methanococcus voltae

    Science.gov (United States)

    Bardy, Sonia L.; Ng, Sandy Y. M.; Carnegie, David S.; Jarrell, Ken F.

    2005-01-01

    Site-directed mutagenesis studies of the signal peptidase of the methanogenic archaeon Methanococcus voltae identified three conserved residues (Ser52, His122, and Asp148) critical for activity. The requirement for one conserved aspartic acid residue distinguishes the archaeal enzyme from both the Escherichia coli and yeast Sec11 enzymes. PMID:15659694

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

    Science.gov (United States)

    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.

  1. Evidence by site-directed mutagenesis that arginine 203 of thermolysin and arginine 717 of neprilysin (neutral endopeptidase) play equivalent critical roles in substrate hydrolysis and inhibitor binding.

    Science.gov (United States)

    Marie-Claire, C; Ruffet, E; Antonczak, S; Beaumont, A; O'Donohue, M; Roques, B P; Fournié-Zaluski, M C

    1997-11-11

    Neprilysin (neutral endopeptidase-24.11, EC 3.4.24.11) is a mammalian zinc-endopeptidase involved in the degradation of biologically active peptides. Although no atomic structure is available for this enzyme, site-directed mutagenesis studies have shown that its active site resembles closely that of the bacterial zinc-endopeptidase, thermolysin (EC 3.4.24.27). One active site residue of thermolysin, Arg-203, is involved in inhibitor binding by forming hydrogen bonds with the carbonyl group of a residue in the P1 position and also participates in a hydrogen bond network involving Asp-170. Sequence alignment data shows that Arg-717 of neprilysin could play a similar role to Arg-203 of thermolysin. This was investigated by site-directed mutagenesis with Arg-203 of thermolysin and Arg-717 of neprilysin being replaced by methionine residues. This led, in both cases, to decreases in kcat/Km values, of 122-fold for neprilysin and 2300-fold for thermolysin, essentially due to changes in kcat. The Ki values of several inhibitors were also increased for the mutated enzymes. In addition, the replacement of Asp-170 of thermolysin by Ala residue resulted in a decrease in kcat/Km of 220-fold. The results, coupled with a molecular modeling study, suggest that Arg-717 of neprilysin corresponds to Arg-203 of thermolysin and that in both enzymes a hydrogen bond network exists, involving His-142, Asp-170, and Arg-203 in thermolysin and His-583, Asp-650, and Arg-717 in neprilysin, which is crucial for hydrolytic activity.

  2. Alteration of Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase Activities by Site-Directed Mutagenesis

    Science.gov (United States)

    Shen, Jennie B.; Ogren, William L.

    1992-01-01

    Site-directed mutagenesis was performed on the 1.6 and 1.9 kilobase spinach (Spinacea oleracea) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase cDNAs, encoding the 41 and 45 kilodalton (kD) isoforms of the enzyme, to create single amino acid changes in the putative ATP-binding site of Rubisco activase (Lys-107, Gln-109, and Ser-112) and in an unrelated cysteine residue (Cys-256). Replacement of Lys-107 with Met produced soluble protein with reduced Rubisco activase and ATPase activities in both isoforms. Substituting Ala or Arg for Lys-107 produced insoluble proteins. Rubisco activase activity increased in the 41-kD isoform when Gln-109 was changed to Glu, but activity in the 45-kD isoform was similar to the wild-type enzyme. ATPase activity in the Glu-109 mutations did not parallel the changes in Rubisco activase activity. Rather, a higher ratio of Rubisco activase to ATPase activity occurred in both isoforms. The mutation of Gln-109 to Lys inactivated Rubisco activase activity. Replacement of Ser-112 with Pro created an inactive protein, whereas attempts to replace Ser-112 with Thr were not successful. The mutation of Cys-256 to Ser in the 45-kD isoform reduced both Rubisco activase and ATPase activities. The results indicate that the two activities of Rubisco activase are not tightly coupled and that variations in photosynthetic efficiency may occur in vivo by replacing the wild-type enzyme with mutant enzymes. ImagesFigure 3 PMID:16668989

  3. Endoglucanase enzyme protein engineering by site-directed mutagenesis to improve the enzymatic properties and its expression in yeast

    Directory of Open Access Journals (Sweden)

    Farnaz Nikzad Jamnani

    2013-11-01

    Full Text Available Introduction: Fossil fuel is an expensive and finite energy source. Therefore, the use of renewable energy and biofuels production has been taken into consideration. One of the most suitable raw materials for biofuels is cellulosic compounds. Only microorganisms that contain cellulose enzymes can decompose cellulose and fungus of Trichodermareesei is the most important producer of this enzyme. Methods: In this study the nucleotide sequence of endoglucanase II, which is the starter of attack to cellulose chains, synthesized from amino acid sequence of this enzyme in fungus T.reesei and based on codon usage in the host; yeast Pichiapastoris. To produce optimized enzyme and to decrease the production time and enzyme price, protein engineering will be used. There are some methods to improve the enzymatic properties like site-directed mutagenesis in which amino-acid replacement occur. In this study two mutations were induced in endoglucanase enzyme gene by PCR in which free syctein positions 169 and 393 were switched to valine and histidine respectively. Then this gene was inserted into the pPinka expression vector and cloned in Escherichia coli. The recombinant plasmids were transferred into P.pastoris competent cells with electroporation, recombinant yeasts were cultured in BMMY medium and induced with methanol. Results: The sequencing of gene proved the induction of the two mutations and the presence of recombinant enzyme was confirmed by dinitrosalicilic acid method and SDS-PAGE. Conclusion: Examination of biochemical properties revealed that the two mutations simultaneously decreased catalytic power, thermal stability and increased the affinity of enzyme and substrate.

  4. Dissecting the Catalytic Mechanism of Betaine-Homocysteine S-Methyltransferase Using Intrinsic Tryptophan Fluorescence and Site-Directed Mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Castro, C.; Gratson, A.A.; Evans, J.C.; Jiracek, J.; Collinsova, M.; Ludwig, M.L.; Garrow, T.A. (ASCR); (UIUC); (Michigan)

    2010-03-05

    Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-({delta}-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind and Met is the last product released. Hcy, Met, and CBHcy bind to the enzyme to form binary complexes with K{sub d} values of 7.9, 6.9, and 0.28 {micro}M, respectively. Binary complexes with Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly to BHMT-Hcy to form ternary complexes with K{sub d} values of 1.1 and 0.73 {micro}M, respectively. Mutation of each of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoes two distinct conformational changes that are reflected in the fluorescence of the enzyme. The first is induced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT, the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacing these residues by site-directed mutagenesis significantly reduces the catalytic efficiency (V{sub max}/K{sub m}) of the enzyme. Replacing Tyr77 with Phe abolishes enzyme activity.

  5. Construction and expression of hepatitis B surface antigen escape variants within the "a" determinant by site directed mutagenesis.

    Science.gov (United States)

    Golsaz Shirazi, Forough; Amiri, Mohammad Mehdi; Mohammadi, Hamed; Bayat, Ali Ahmad; Roohi, Azam; Khoshnoodi, Jalal; Zarnani, Amir Hassan; Jeddi-Tehrani, Mahmood; Kardar, Gholam Ali; Shokri, Fazel

    2013-09-01

    The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect the performance of HBsAg assays. To construct clinically relevant recombinant mutant forms of HBsAg and assessment of their reactivity with anti-HBs monoclonal antibodies (MAbs). Wild type (wt) and mutant (mt) HBsAg genes were constructed by site directed mutagenesis and SEOing PCR. The amplified genes were inserted into pCMV6-neo plasmid and transfected in CHO cell line. The expression of wt- and mtHBsAg was assessed by commercial ELISA assays and stable cells were established and cloned by limiting dilution. The recombinant mutants were further characterized using a panel of anti-HBs monoclonal antibodies (MAbs) and the pattern of their reactivity was assessed by ELISA. Ten HBsAg mutants having single mutation within the "a" determinant including P120E, T123N, Q129H, M133L, K141E, P142S, D144A, G145R, N146S and C147S together with a wt form were successfully constructed and expressed in CHO cells. Reactivity of anti-HBs MAbs with mtHBsAgs displayed different patterns. The effect of mutations on antibody binding differed depending on the amino acid involved and its location within the ''a'' determinant. Mutation at amino acids 123 and 145 resulted in either complete loss or significant reduction of binding to all anti-HBs MAbs. Our panel of mtHBsAgs is a valuable tool for assessment of the antibody response to HBV escape mutants and may have substantial implications in HBV immunological diagnostics.

  6. Transcarboxylase (TC): demonstration by site-directed mutagenesis that methionines at the biotin site are essential for catalysis

    International Nuclear Information System (INIS)

    Wood, H.G.; Shenoy, B.C.; Kumar, G.K.; Paranjape, S.; Murtif, V.; Samols, D.

    1987-01-01

    All biotin enzymes that have thus far been sequenced contain a conserved region ALA MET BCT MET. Two possible roles of the conserved region are (i) for recognition of the specific lysine of the enzyme that is to be biotinated posttranslationally by the synthetase or (ii) for activation of the biotin to function as a carboxyl carrier. The BCT of TC is at residue 89 of the 1.3S subunit. By site-directed mutagenesis, single amino acid substitutions have been made giving LEU 88, THR 88 and LEU 90 and these mutant subunits have been expressed in E. coli and isolated. Catalysis by TC involves Partial Reactions: (1) - 00 14 CCH 2 COCOO - + 1.3S biotin pyruvate + 1.3S biotin-COO - catalyzed by the 5S subunit (2) 14 CH 3 CH( 14 COO - )COSCoA + 1.3S biotin CH 3 CH 2 COSCoA + 1.3S biotin- 14 COO - , catalyzed by the 12S subunit. The mutant subunits LEU 88 and THR 88 are inactive in Reaction 1. In Reaction 2, they are 8% as active as the 1.3S wild type. At 10 times the concentration of the wild type, they are 40% as active. The LEU 90 subunit is about 40% as active as wild type in both Reactions 1 and 2. Thus, the two METS are functionally not equivalent. What their catalytic roles are remains to be determined. Shenoy et al. have shown these modifications do not effect the synthetase reaction

  7. High-resolution mapping of the HyHEL-10 epitope of chicken lysozyme by site-directed mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Kam-Morgan, L.N.; Taylor, M.G.; Kirsch, J.F. (Lawrence Berkeley Laboratory, CA (United States)); Smith-Gill, S.J. (National Institutes of Health, Bethesda, MD (United States)); Wilson, A.C.

    1993-05-01

    The complex formed between hen egg white lysozyme (HEL) and the monoclonal antibody HyHEL-10 Fab fragment has an interface composed of van der Waals interactions, hydrogen bonds, and a single ion pair. The antibody overlaps part of the active site cleft. Putative critical residues within the epitope region of HEL, identified from the x-ray crystallographic structure of the complex, were replaced by site-directed mutagenesis to probe their relative importance in determining affinity of the antibody for HEL. Twenty single mutations of HEL at three contact residues (Arg-21[sub HEL], Asp-101[sub HEL], and Gly-102[sub HEL]) and at a partially buried residue (Asn-19[sub HEL]) in the epitope were made, and the effects on the free energies of dissociation were measured. A correlation between increased amino acid side-chain volume and reduced affinity for HELs with mutations at position 101 was observed. The D101G[sub HEL] mutant is bound to HyHEL-10 as tightly as wild-type enzyme, but the [delta][delta]G[sub dissoc] is increased by about 2.2 kcal (9.2 kJ)/mol for the larger residues in this position. HEL variants with lysine or histidine replacements for arginine at position 21 are bound 1.4-2.7 times more tightly than those with neutral or negatively charged amino acids in this position. These exhibit 1/40 the affinity for HyHEL-10 Fab compared with wild type. There is no side-chain volume correlation with [delta][delta]G[sub dissoc] at position 21. Although Gly-102[sub HEL] and Asn-19[sub HEL] are in the epitope, replacements at these positions have no effect on the affinity of HEL for the antibody. 34 refs., 2 figs., 3 tabs.

  8. SiteFind: A software tool for introducing a restriction site as a marker for successful site-directed mutagenesis

    Directory of Open Access Journals (Sweden)

    Evans Paul M

    2005-12-01

    Full Text Available Abstract Background Site-directed mutagenesis is a widely-used technique for introducing mutations into a particular DNA sequence, often with the goal of creating a point mutation in the corresponding amino acid sequence but otherwise leaving the overall sequence undisturbed. However, this method provides no means for verifying its success other than sequencing the putative mutant construct: This can quickly become an expensive method for screening for successful mutations. An alternative to sequencing is to simultaneously introduce a restriction site near the point mutation in manner such that the restriction site has no effect on the translated amino acid sequence. Thus, the novel restriction site can be used as a marker for successful mutation which can be quickly and easily assessed. However, finding a restriction site that does not disturb the corresponding amino acid sequence is a time-consuming task even for experienced researchers. A fast and easy to use computer program is needed for this task. Results We wrote a computer program, called SiteFind, to help us design a restriction site within the mutation primers without changing the peptide sequence. Because of the redundancy of genetic code, a given peptide can be encoded by many different DNA sequences. Since the list of possible restriction sites for a given DNA sequence is not always obvious, SiteFind automates this task. The number of possible sequences a computer program must search through increases exponentially as the sequence length increases. SiteFind uses a novel "moving window" algorithm to reduce the number of possible sequences to be searched to a manageable level. The user enters a nucleotide sequence, specifies what amino acid residues should be changed in the mutation, and SiteFind generates a list of possible restriction sites and what nucleotides must be changed to introduce that site. As a demonstration of its use, we successfully generated a single point mutation

  9. Evidence by site-directed mutagenesis that arginine 203 of thermolysin and arginine 717 of neprilysin (neutral endopeptidase) play equivalent critical roles in substrate hydrolysis and inhibitor binding.

    OpenAIRE

    Marie-Claire, Cynthia; Ruffet, Emmanuel; Antonczak, Serge; Beaumont, Ann; O'Donohue, Michael; Roques, Bernard,; Fournié-Zaluski, Marie-Claude

    1997-01-01

    International audience; Neprilysin (neutral endopeptidase-24.11, EC 3.4.24.11) is a mammalian zinc-endopeptidase involved in the degradation of biologically active peptides. Although no atomic structure is available for this enzyme, site-directed mutagenesis studies have shown that its active site resembles closely that of the bacterial zinc-endopeptidase, thermolysin (EC 3.4.24.27). One active site residue of thermolysin, Arg-203, is involved in inhibitor binding by forming hydrogen bonds wi...

  10. Structure-guided approach identifies a novel class of HIV-1 ribonuclease H inhibitors: binding mode insights through magnesium complexation and site-directed mutagenesis studies

    DEFF Research Database (Denmark)

    Poongavanam, Vasanthanathan; Corona, Angela; Steinmann, Casper

    2018-01-01

    with innovative mechanisms. The HIV-1 reverse transcriptase (RT) associated ribonuclease H (RNase H) is the only viral encoded enzyme that still lacks an efficient inhibitor despite the fact that it is a well-validated target whose functional abrogation compromises viral infectivity. Identification of new drugs...... inhibited the RNase H function below 100 μM with three hits exhibiting IC50 values mode of inhibition. Site-directed mutagenesis studies provide valuable insight into the binding mode of newly...

  11. New insights into the QuikChangeTM process guide the use of Phusion DNA polymerase for site-directed mutagenesis

    Science.gov (United States)

    Xia, Yongzhen; Chu, Wenqiao; Qi, Qingsheng; Xun, Luying

    2015-01-01

    The QuikChangeTM site-directed mutagenesis method is popular but imperfect. An improvement by using partially overlapping primers has been reported several times; however, it is incompatible with the proposed mechanism. The QuikChangeTM method using complementary primers is proposed to linearly amplify a target plasmid with the products annealing to produce double-stranded DNA molecules with 5′-overhangs. The overhang annealing is supposed to form circular plasmids with staggered breaks, which can be repaired in Escherichia coli after transformation. Here, we demonstrated that the PCR enzyme fills the 5′-overhangs in the early cycles, and the product is then used as the template for exponential amplification. The linear DNA molecules with homologous ends are joined to generate the plasmid with the desired mutations through homologous recombination in E. coli. The correct understanding is important to method improvements, guiding us to use partially overlapping primers and Phusion DNA polymerase for site-directed mutagenesis. Phusion did not amplify a plasmid with complementary primers but used partially overlapping primers to amplify the plasmid, producing linear DNA molecules with homologous ends for site-directed mutagenesis. PMID:25399421

  12. Probing substrate binding to Metallo-β-Lactamase L1 from Stenotrophomonas maltophilia by using site-directed mutagenesis

    Directory of Open Access Journals (Sweden)

    Yates Robert B

    2002-02-01

    Full Text Available Abstract Background The metallo-β-lactamases are Zn(II-containing enzymes that hydrolyze the β-lactam bond in penicillins, cephalosporins, and carbapenems and are involved in bacterial antibiotic resistance. There are at least 20 distinct organisms that produce a metallo-β-lactamase, and these enzymes have been extensively studied using X-ray crystallographic, computational, kinetic, and inhibition studies; however, much is still unknown about how substrates bind and the catalytic mechanism. In an effort to probe substrate binding to metallo-β-lactamase L1 from Stenotrophomonas maltophilia, nine site-directed mutants of L1 were prepared and characterized using metal analyses, CD spectroscopy, and pre-steady state and steady state kinetics. Results Site-directed mutations were generated of amino acids previously predicted to be important in substrate binding. Steady-state kinetic studies using the mutant enzymes and 9 different substrates demonstrated varying Km and kcat values for the different enzymes and substrates and that no direct correlation between Km and the effect of the mutation on substrate binding could be drawn. Stopped-flow fluorescence studies using nitrocefin as the substrate showed that only the S224D and Y228A mutants exhibited weaker nitrocefin binding. Conclusions The data presented herein indicate that Ser224, Ile164, Phe158, Tyr228, and Asn233 are not essential for tight binding of substrate to metallo-β-lactamase L1. The results in this work also show that Km values are not reliable for showing substrate binding, and there is no correlation between substrate binding and the amount of reaction intermediate formed during the reaction. This work represents the first experimental testing of one of the computational models of the metallo-β-lactamases.

  13. Site-directed mutagenesis, kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp. YM55-1.

    Science.gov (United States)

    Puthan Veetil, Vinod; Raj, Hans; Quax, Wim J; Janssen, Dick B; Poelarends, Gerrit J

    2009-06-01

    Aspartate ammonia lyases (also referred to as aspartases) catalyze the reversible deamination of L-aspartate to yield fumarate and ammonia. In the proposed mechanism for these enzymes, an active site base abstracts a proton from C3 of L-aspartate to form an enzyme-stabilized enediolate intermediate. Ketonization of this intermediate eliminates ammonia and yields the product, fumarate. Although two crystal structures of aspartases have been determined, details of the catalytic mechanism have not yet been elucidated. In the present study, eight active site residues (Thr101, Ser140, Thr141, Asn142, Thr187, His188, Lys324 and Asn326) were mutated in the structurally characterized aspartase (AspB) from Bacillus sp. YM55-1. On the basis of a model of the complex in which L-aspartate was docked manually into the active site of AspB, the residues responsible for binding the amino group of L-aspartate were predicted to be Thr101, Asn142 and His188. This postulate is supported by the mutagenesis studies: mutations at these positions resulted in mutant enzymes with reduced activity and significant increases in the K(m) for L-aspartate. Studies of the pH dependence of the kinetic parameters of AspB revealed that a basic group with a pK(a) of approximately 7 and an acidic group with a pK(a) of approximately 10 are essential for catalysis. His188 does not play the typical role of active site base or acid because the H188A mutant retained significant activity and displayed an unchanged pH-rate profile compared to that of wild-type AspB. Mutation of Ser140 and Thr141 and kinetic analysis of the mutant enzymes revealed that these residues are most likely involved in substrate binding and in stabilizing the enediolate intermediate. Mutagenesis studies corroborate the essential role of Lys324 because all mutations at this position resulted in mutant enzymes that were completely inactive. The substrate-binding model and kinetic analysis of mutant enzymes suggest that Thr187 and Asn326

  14. From Green to Blue: Site-Directed Mutagenesis of the Green Fluorescent Protein to Teach Protein Structure-Function Relationships

    Science.gov (United States)

    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. Delineation of the structural and functional role of Arg111 in GSTU4-4 from Glycine max by chemical modification and site-directed mutagenesis.

    Science.gov (United States)

    Labrou, Nikolaos E; Muharram, Magdy Mohamed; Abdelkader, Maged Saad

    2016-10-01

    The structural and functional role of Arg111 in GSTU4-4 from Glycine max (GmGSTU4-4) was studied by chemical modification followed by site-directed mutagenesis. The arginine-specific reagent 2,3-butanedione (BTD) inactivates the enzyme in borate buffer at pH8.0, with pseudo-first-order saturation kinetics. The rate of inactivation exhibited a non-linear dependence on the concentration of BTD which can be described by reversible binding of reagent to the enzyme (KD 81.2±9.2mM) prior to the irreversible reaction, with maximum rate constants of 0.18±0.01min(-1). Protection from inactivation was afforded by substrate analogues demonstrating the specificity of the reaction. Structural analysis suggested that the modified residue is Arg111, which was confirmed by protein chemistry experiments. Site-directed mutagenesis was used in dissecting the role of Arg111 in substrate binding, specificity and catalytic mechanism. The mutant Arg111Ala enzyme exhibited unchanged Km value for GSH but showed reduced affinity for the xenobiotic substrates, higher kcat and specific activities towards aromatic substrates and lower specific activities towards aliphatic substrates. The biological significance of the specific modification of Arg111 by dicarbonyl compounds and the role of Arg111 as a target for engineering xenobiotic substrate specificity were discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Improved thermostability and enzyme activity of a recombinant phyA mutant phytase from Aspergillus niger N25 by directed evolution and site-directed mutagenesis.

    Science.gov (United States)

    Tang, Zizhong; Jin, Weiqiong; Sun, Rong; Liao, Yan; Zhen, Tianrun; Chen, Hui; Wu, Qi; Gou, Lin; Li, Chenlei

    2018-01-01

    We previously constructed three recombinant phyA mutant strains (PP-NP m -8, PP-NP ep -6A and I44E/T252R-PhyA), showing improved catalytic efficiency or thermostability of Aspergillus niger N25 phytase, by error-prone PCR or site-directed mutagenesis. In this study, directed evolution and site-directed mutagenesis were further applied to improve the modified phytase properties. After one-round error-prone PCR for phytase gene of PP-NP ep -6A, a single transformant, T195L/Q368E/F376Y, was obtained with the significant improvements in catalytic efficiency and thermostability. The phytase gene of T195L/Q368E/F376Y, combined with the previous mutant phytase genes of PP-NP ep -6A, PP-NP m -8 and I44E/T252R-PhyA, was then sequentially modified by DNA shuffling. Three genetically engineered strains with desirable properties were then obtained, namedQ172R, Q172R/K432R andQ368E/K432R. Among them, Q172R/K432R showed the highest thermostability with the longest half-life and the greatest remaining phytase activity after heat treatment, while Q368E/K432R showed the highest catalytic activity. Five substitutions (Q172R, T195L, Q368E, F376Y, K432R) identified from random mutagenesis were added sequentially to the phytase gene of PP-NP ep -6A to investigate how the mutant sites influence the properties of phytase. Characterization and structural analysis demonstrated that these mutations could produce cumulative or synergistic improvements in thermostability or catalytic efficiency of phytase. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: [Fe-S] cluster-driven protein rearrangement

    International Nuclear Information System (INIS)

    Martin, A.E.; Burgess, B.K.; Stout, C.D.; Cash, V.L.; Dean, D.R.; Jensen, G.M.; Stephens, P.J.

    1990-01-01

    Azotobacter vinelandii ferredoxin I is a small protein that contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. Recently the x-ray crystal structure has been redetermined and the fdxA gene, which encodes the protein, has been cloned and sequenced. Here the authors report the site-directed mutation of Cys-20, which is a ligand of the [4Fe-4S] cluster in the native protein, to alanine and the characterization of the protein product by x-ray crystallographic and spectroscopic methods. The data show that the mutant protein again contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. The new [4Fe-4S] cluster obtains its fourth ligand from Cys-24, a free cysteine in the native structure. The formation of this [4Fe-4S] cluster drives rearrangement of the protein structure

  18. Catalytic roles of lysines (K9, K27, K31) in the N-terminal domain in human adenylate kinase by random site-directed mutagenesis.

    Science.gov (United States)

    Ayabe, T; Park, S K; Takenaka, H; Sumida, M; Uesugi, S; Takenaka, O; Hamada, M

    1996-11-01

    To elucidate lysine residues in the N-terminal domain of human cytosolic adenylate kinase (hAK1, EC 2.7.4.3), random site-directed mutagenesis of K9, K27, and K31 residues was performed, and six mutants were analyzed by steady-state kinetics. K9 residue may play an important role in catalysis by interacting with AMP2-. K27 and K31 residues appear to play a functional role in catalysis by interacting with MgATP2-. In human AK, the epsilon-amino group in the side chain of these lysine residues would be essential for phosphoryl transfer between MgATP2- and AMP2- during transition state.

  19. Site-Directed Mutagenesis Study Revealed Three Important Residues in Hc-DAF-22, a Key Enzyme Regulating Diapause of Haemonchus contortus

    Directory of Open Access Journals (Sweden)

    Yan Huang

    2017-11-01

    Full Text Available Haemonchus contortus (H. contortus is one of the most important parasites of small ruminants, especially goats and sheep. The complex life cycle of this nematode is a main obstacle for the control and prevention of haemonchosis. So far, a special form of arrested development called diapause different from the dauer stage in Caenorhabditis elegans (C. elegans has been found in many parasitic nematodes. In our previous study, we have characterized a novel gene Hc-daf-22 from H. contortus sharing high homology with Ce-daf-22 and functional analysis showed this gene has similar biological function with Ce-daf-22. In this study, Hc-daf-22 mutants were constructed using site-directed mutagenesis, and carried out rescue experiments, RNA interference (RNAi experiments and in vitro enzyme activity analysis with the mutants to further explore the precise function site of Hc-DAF-22. The results showed that Hc-daf-22 mutants could be expressed in the rescued ok693 worms and the expression positions were mainly in the intestine which was identical with that of Hc-daf-22 rescued worms. Through lipid staining we found that Hc-daf-22 could rescue daf-22 mutant (ok693 from the fatty acid metabolism deficiency while Hc-daf-22 mutants failed. Brood size and body length analyses in rescue experiment along with body length and life span analyses in RNAi experiment elucidated that Hc-daf-22 resembled Ce-daf-22 in effecting the development and capacity of C. elegans and mutants impaired the function of Hc-daf-22. Together with the protease activity assay, this research revealed three important active resides 84C/299H/349H in Hc-DAF-22 by site-directed mutagenesis.

  20. Site-Directed Mutagenesis Study Revealed Three Important Residues in Hc-DAF-22, a Key Enzyme Regulating Diapause of Haemonchus contortus.

    Science.gov (United States)

    Huang, Yan; Zheng, Xiuping; Zhang, Hongli; Ding, Haojie; Guo, Xiaolu; Yang, Yi; Chen, Xueqiu; Zhou, Qianjin; Du, Aifang

    2017-01-01

    Haemonchus contortus ( H. contortus ) is one of the most important parasites of small ruminants, especially goats and sheep. The complex life cycle of this nematode is a main obstacle for the control and prevention of haemonchosis. So far, a special form of arrested development called diapause different from the dauer stage in Caenorhabditis elegans ( C. elegans ) has been found in many parasitic nematodes. In our previous study, we have characterized a novel gene Hc-daf-22 from H. contortus sharing high homology with Ce-daf-22 and functional analysis showed this gene has similar biological function with Ce-daf-22 . In this study, Hc-daf-22 mutants were constructed using site-directed mutagenesis, and carried out rescue experiments, RNA interference (RNAi) experiments and in vitro enzyme activity analysis with the mutants to further explore the precise function site of Hc-DAF-22. The results showed that Hc-daf-22 mutants could be expressed in the rescued ok693 worms and the expression positions were mainly in the intestine which was identical with that of Hc-daf-22 rescued worms. Through lipid staining we found that Hc-daf-22 could rescue daf-22 mutant ( ok693 ) from the fatty acid metabolism deficiency while Hc-daf-22 mutants failed. Brood size and body length analyses in rescue experiment along with body length and life span analyses in RNAi experiment elucidated that Hc-daf-22 resembled Ce-daf-22 in effecting the development and capacity of C. elegans and mutants impaired the function of Hc-daf-22 . Together with the protease activity assay, this research revealed three important active resides 84C/299H/349H in Hc-DAF-22 by site-directed mutagenesis.

  1. Gene knockouts, in vivo site-directed mutagenesis and other modifications using the delitto perfetto system in Saccharomyces cerevisiae.

    Science.gov (United States)

    Stuckey, Samantha; Storici, Francesca

    2013-01-01

    (Storici et al., 1999; Sauer, 1987). The presence of such scars can threaten the genomic stability of the strain and/or limit the number of successive genetic manipulations for that strain. Here, we describe the delitto perfetto approach for in vivo mutagenesis that combines the practicality of a general selection system with the versatility of synthetic oligonucleotides for targeting (Storici et al., 2001). It provides for generation of gene knockouts and almost any sort of mutation and genome rearrangement via HR. The delitto perfetto in vivo mutagenesis technique is designed for efficient and precise manipulation of yeast strains in a two-step process spanning ~2 weeks. Here, we present the theory and procedures of the delitto perfetto technique. © 2013 Elsevier Inc. All rights reserved.

  2. Site-directed mutagenesis studies on the uridine monophosphate binding sites of feedback inhibition in carbamoyl phosphate synthetase and effects on cytidine production by Bacillus amyloliquefaciens.

    Science.gov (United States)

    Fang, Haitian; Liu, Huiyan; Chen, Ning; Zhang, Chenglin; Xie, Xixian; Xu, Qingyang

    2013-06-01

    A major problem when pyrimidine de novo biosynthesis is used for cytidine production is the existence of many negative regulatory factors. Cytidine biosynthesis in Bacillus amyloliquefaciens proceeds via a pathway that is controlled by uridine monophosphate (UMP) through feedback inhibition of carbamoyl phosphate synthetase (CPS), the enzyme that converts CO2, NH3, and glutamine to carbamoyl phosphate. In this study, the gene carB encoding the large subunit of CPS from B. amyloliquefaciens CYT1 was site directed, and the UMP binding sites of feedback inhibition in Bam-CPS are described. The residues Thr-941, Thr-970, and Lys-986 in CPS from B. amyloliquefaciens were subjected to site-directed mutagenesis to alter UMP's feedback inhibition of CPS. To find feedback-resistant B. amyloliquefaciens, the influence of the T941F, T970A, K986I, T941F/K986I, and T941F/T970A/K986I mutations on CPS enzymatic properties was studied. The recombinant B. amyloliquefaciens with mutated T941F/K986I and T941F/T970A/K986I CPS showed a 3.7- and 5.7-fold increase, respectively, in cytidine production in comparison with the control expressing wild-type CPS, which was more suitable for further application of the cytidine synthesis. To a certain extent, the 5 mutations were found to release the enzyme from UMP inhibition and to improve B. amyloliquefaciens cytidine-producing strains.

  3. Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies

    Directory of Open Access Journals (Sweden)

    Edelheit Oded

    2009-06-01

    Full Text Available Abstract Background In protein engineering, site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions. In a widely used method, mutations are generated by PCR using a pair of oligonucleotide primers designed with mismatching nucleotides at the center of the primers. In this method, primer-primer annealing may prevent cloning of mutant cDNAs. To circumvent this problem we developed an alternative procedure that does not use forward-reverse primer pair in the same reaction. Results In initial studies we used a double-primer PCR mutagenesis protocol, but sequencing of products showed tandem repeats of primer in cloned DNA. We developed an alternative method that starts with two Single-Primer Reactions IN Parallel using high-fidelity Pwo DNA polymerase. Thus, we call the method with the acronym SPRINP. The SPRINP reactions are then combined, denatured at 95°C, and slowly cooled, promoting random annealing of the parental DNA and the newly synthesized strands. The products are digested with DpnI that digests methylated parental strands, and then transformed into E. coli. Using this method we generated >40 mutants in cDNAs coding for human Epithelial Na+ Channel (ENaC subunits. The method has been tested for 1–3 bp codon mutation and insertion of a 27 bp epitope tag into cDNAs. Conclusion The SPRINP mutagenesis protocol yields mutants reliably and with high fidelity. The use of a single primer in each amplification reaction increases the probability of success of primers relative to previous methods employing a forward and reverse primer pair in the same reaction.

  4. Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies.

    Science.gov (United States)

    Edelheit, Oded; Hanukoglu, Aaron; Hanukoglu, Israel

    2009-06-30

    In protein engineering, site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions. In a widely used method, mutations are generated by PCR using a pair of oligonucleotide primers designed with mismatching nucleotides at the center of the primers. In this method, primer-primer annealing may prevent cloning of mutant cDNAs. To circumvent this problem we developed an alternative procedure that does not use forward-reverse primer pair in the same reaction. In initial studies we used a double-primer PCR mutagenesis protocol, but sequencing of products showed tandem repeats of primer in cloned DNA. We developed an alternative method that starts with two Single-Primer Reactions IN Parallel using high-fidelity Pwo DNA polymerase. Thus, we call the method with the acronym SPRINP. The SPRINP reactions are then combined, denatured at 95 degrees C, and slowly cooled, promoting random annealing of the parental DNA and the newly synthesized strands. The products are digested with DpnI that digests methylated parental strands, and then transformed into E. coli. Using this method we generated >40 mutants in cDNAs coding for human Epithelial Na+ Channel (ENaC) subunits. The method has been tested for 1-3 bp codon mutation and insertion of a 27 bp epitope tag into cDNAs. The SPRINP mutagenesis protocol yields mutants reliably and with high fidelity. The use of a single primer in each amplification reaction increases the probability of success of primers relative to previous methods employing a forward and reverse primer pair in the same reaction.

  5. Mapping the heparin-binding site of the osteoinductive protein NELL1 by site-directed mutagenesis.

    Science.gov (United States)

    Takahashi, Kaneyoshi; Imai, Arisa; Iijima, Masumi; Yoshimoto, Nobuo; Maturana, Andrés D; Kuroda, Shun'ichi; Niimi, Tomoaki

    2015-12-21

    Neural epidermal growth factor-like (NEL)-like 1 (NELL1) is a secretory osteogenic protein comprising an N-terminal thrombospondin-1-like (TSPN) domain, four von Willebrand factor type C domains, and six epidermal growth factor-like repeats. NELL1 shows heparin-binding activity; however, the biological significance remains to be explored. In this report, we demonstrate that NELL1 binds to cell surface proteoglycans through its TSPN domain. Major heparin-binding sites were identified on the three-dimensional structural model of the TSPN domain of NELL1. Mutant analysis of the heparin-binding sites indicated that the heparin-binding activity of the TSPN domain is involved in interaction of NELL1 with cell surface proteoglycans. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  6. Enhancement of the catalytic activity of ferulic acid decarboxylase from Enterobacter sp. Px6-4 through random and site-directed mutagenesis.

    Science.gov (United States)

    Lee, Hyunji; Park, Jiyoung; Jung, Chaewon; Han, Dongfei; Seo, Jiyoung; Ahn, Joong-Hoon; Chong, Youhoon; Hur, Hor-Gil

    2015-11-01

    The enzyme ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4 catalyzes the decarboxylation reaction of lignin monomers and phenolic compounds such as p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, that is, 4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol, respectively. Among various ferulic acid decarboxylase enzymes, we chose the FADase from Enterobacter sp. Px6-4, whose crystal structure is known, and produced mutants to enhance its catalytic activity by random and site-directed mutagenesis. After three rounds of sequential mutations, FADase(F95L/D112N/V151I) showed approximately 34-fold higher catalytic activity than wild-type for the production of 4-vinylguaiacol from ferulic acid. Docking analyses suggested that the increased activity of FADase(F95L/D112N/V151I) could be due to formation of compact active site compared with that of the wild-type FADase. Considering the amount of phenolic compounds such as lignin monomers in the biomass components, successfully bioengineered FADase(F95L/D112N/V151I) from Enterobacter sp. Px6-4 could provide an ecofriendly biocatalytic tool for producing diverse styrene derivatives from biomass.

  7. Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues.

    Science.gov (United States)

    Kobayashi, M; Izui, H; Nagasawa, T; Yamada, H

    1993-01-01

    Indole-3-acetic acid is the major auxin in most plants. In Cruciferae, including Brassicaceae, indole-3-acetic acid is synthesized from indole-3-acetonitrile by nitrilase, after indole-3-acetonitrile is formed from tryptophan via indole-3-acetaldoxime or indole glycosinolates as the intermediate. We cloned and sequenced the gene for nitrilase (EC 3.5.5.1), which catalyzes the hydrolysis of indole-3-acetonitrile to indole-3-acetic acid, from Alcaligenes faecalis JM3. The amino acid sequence deduced from the nucleotide sequence of the nitrilase gene shows 34.7% identity with that of Klebsiella ozaenae nitrilase. A DNA clone containing the nitrilase gene expressed the active enzyme in Escherichia coli with excellent yield. Among five cysteine residues (Cys-40, Cys-115, Cys-162, Cys-163, and Cys-218) in the Alcaligenes nitrilase, only Cys-163 was conserved at the corresponding position in the Klebsiella nitrilase. Two mutant enzymes, in which Cys-162 and Cys-163 were replaced with Asn and Ala, respectively, were constructed by site-directed mutagenesis. A 35% increase of the specific activity and a large reduction of the Km for thiophene-2-acetonitrile (which was used as a standard substrate for the nitrilase) were observed in the Cys-162-->Asn mutant enzyme. The Cys-163-->Ala mutation resulted in complete loss of nitrilase activity, clearly indicating that Cys-163 is crucial for the activity and Cys-162 could not provide the catalytic function of Cys-163. Images PMID:8419930

  8. Key amino acid associated with acephate detoxification by Cydia pomonella carboxylesterase based on molecular dynamics with alanine scanning and site-directed mutagenesis.

    Science.gov (United States)

    Yang, Xue Qing; Liu, Ji Yuan; Li, Xian Chun; Chen, Mao Hua; Zhang, Ya Lin

    2014-05-27

    Insecticide-detoxifying carboxylesterase (CE) gene CpCE-1 was cloned from Cydia pomonella. Molecular dynamics (MD) simulation and computational alanine scanning (CAS) indicate that Asn 232 in CpCE-1 constitutes an approximate binding hot-spot with a binding free energy difference (ΔΔGbind) value of 3.66 kcal/mol. The catalytic efficiency (kcat/km) of N232A declined dramatically, and the half inhibitory concentrations (IC50) value increased by more than 230-fold. Metabolism assay in vitro reveals that the acephate could be metabolized by wild CpCE-1, whereas N232A mutation is unable to metabolize the acephate, which suggests that the hot-spot Asn 232 is a crucial residue for acephate metabolism. Mutation detection suggests that low frequency of Asn 232 replacement occurred in Europe field strains. Our MD, CAS, site-directed mutagenesis, and metabolism studies introduce a new amino acid residue Asn 232 involved in the metabolism of the acephate with CpCE-1, and this method is reliable in insecticide resistance mechanism research and prediction of key amino acids in a protein which is associated with specific physiological and biochemical functions.

  9. Site-directed Mutagenesis (Y52E) of POLH Affects Its Ability to Bypass Ultraviolet-induced DNA Lesions in HaCaT Cells.

    Science.gov (United States)

    Luo, C; Chen, Z; He, Q; Cao, K; Wang, S; Liu, J; Liu, R; Zhou, J

    2014-08-01

    DNA polymerase eta (Pol η) is one of several Y family trans-lesion synthesis (TLS) polymerases in humans and plays an important role in maintaining genome stability after ultraviolet (UV) irradiation, as it carries out error-free TLS at sites of UV-induced lesions. We performed site-directed mutagenesis of human polymerase η gene (Y52E), confirmed by sequencing, then cloned wild-type mutant and POLH genes into the eukaryotic vector pEGFP-N1. After transfecting wild-type and mutant plasmids into HaCaT keratinocytes, we tested for UV induced cis-syn cyclobutane pyrimidine dimer (CPDs) DNA lesions, and analysed cellular viability by MTT cell proliferation assay. The results showed that CPD levels decreased both with empty vector control (EVC), wild-type POLH, and Y52E-POLH over 48 hours post UV irradiation with 0.1 mW/cm2 UVB for 15 minutes (p = 0.025). The rate in CPD reduction of mutant POLH was less than in wild-type POLH. Cell viabilities of all three groups increased over 48 hours after UV irradiation, with the increased rate in the wild-type being higher than for mutant protein (p = 0.046). We conclude that Y52E POLH has reduced capacity to bypass UV induced DNA lesions in HaCaT cells.

  10. Novel induced mlo mutant alleles in combination with site-directed mutagenesis reveal functionally important domains in the heptahelical barley Mlo protein

    Directory of Open Access Journals (Sweden)

    Piffanelli Pietro

    2010-02-01

    Full Text Available Abstract Background Recessively inherited natural and induced mutations in the barley Mlo gene confer durable broad-spectrum resistance against the powdery mildew pathogen, Blumeria graminis f.sp. hordei. Mlo codes for a member of a plant-specific family of polytopic integral membrane proteins with unknown biochemical activity. Resistant barley mlo mutant alleles identify amino acid residues that are critical for Mlo function in the context of powdery mildew susceptibility. Results We molecularly analyzed a novel set of induced barley mlo mutants and used site-directed mutagenesis in combination with transient gene expression to unravel novel amino acid residues of functional significance. We integrate these results with previous findings to map functionally important regions of the heptahelical Mlo protein. Our data reveal the second and third cytoplasmic loop as being particularly sensitive to functional impediment by mutational perturbation, suggesting that these regions are critical for the susceptibility-conferring activity of the Mlo protein. In contrast, only mutations in the second but not the third cytoplasmic loop appear to trigger the Endoplasmic Reticulum-localized quality control machinery that ensures the biogenesis of properly folded membrane proteins. Conclusion Our findings identify functionally important regions of the polytopic barley Mlo protein and reveal the differential sensitivity of individual protein domains to cellular quality control.

  11. A simple, flexible and efficient PCR-fusion/Gateway cloning procedure for gene fusion, site-directed mutagenesis, short sequence insertion and domain deletions and swaps

    Directory of Open Access Journals (Sweden)

    Etchells J Peter

    2009-10-01

    Full Text Available Abstract Background The progress and completion of various plant genome sequencing projects has paved the way for diverse functional genomic studies that involve cloning, modification and subsequent expression of target genes. This requires flexible and efficient procedures for generating binary vectors containing: gene fusions, variants from site-directed mutagenesis, addition of protein tags together with domain swaps and deletions. Furthermore, efficient cloning procedures, ideally high throughput, are essential for pyramiding of multiple gene constructs. Results Here, we present a simple, flexible and efficient PCR-fusion/Gateway cloning procedure for construction of binary vectors for a range of gene fusions or variants with single or multiple nucleotide substitutions, short sequence insertions, domain deletions and swaps. Results from selected applications of the procedure which include ORF fusion, introduction of Cys>Ser mutations, insertion of StrepII tag sequence and domain swaps for Arabidopsis secondary cell wall AtCesA genes are demonstrated. Conclusion The PCR-fusion/Gateway cloning procedure described provides an elegant, simple and efficient solution for a wide range of diverse and complicated cloning tasks. Through streamlined cloning of sets of gene fusions and modification variants into binary vectors for systematic functional studies of gene families, our method allows for efficient utilization of the growing sequence and expression data.

  12. Enhancement in catalytic activity of Aspergillus niger XynB by selective site-directed mutagenesis of active site amino acids.

    Science.gov (United States)

    Wu, Xiuyun; Tian, Zhennan; Jiang, Xukai; Zhang, Qun; Wang, Lushan

    2018-01-01

    XynB from Aspergillus niger ATCC1015 (AnXynB) is a mesophilic glycoside hydrolase (GH) family 11 xylanase which holds great potentials in a wide variety of industrial applications. In the present study, the catalytic activity and stability of AnXynB were improved by a combination of computational and experimental approaches. Virtual mutation and molecular dynamics simulations indicated that the introduction of Glu and Asn altered the interaction network at the - 3 subsite. Interestingly, the double mutant S41N/T43E displayed 72% increase in catalytic activity when compared to the wild type (WT). In addition, it also showed a better thermostability than the WT enzyme. Kinetic determination of the T43E and S41N/T43E mutants suggested that the higher xylanase activity is probably due to the increasing binding affinity of enzyme and substrate. Consequently, the enzyme activity and thermostability of AnXynB was both increased by selective site-directed mutagenesis at the - 3 subsite of its active site architecture which provides a good example for a successfully engineered enzyme for potential industrial application. Moreover, the molecular evolution approach adopted in this study led to the design of a library of sequences that captures a meaningful functional diversity in a limited number of protein variants.

  13. Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods.

    Science.gov (United States)

    Cristaldi, Julio C; Gómez, María C; González, Pablo J; Ferroni, Felix M; Dalosto, Sergio D; Rizzi, Alberto C; Rivas, María G; Brondino, Carlos D

    2018-03-01

    The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (k cat =3.9(4) s -1 ) lower than that of wt SmNir (k cat =240(50) s -1 ). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the N δ1 H…OC hydrogen bond that partially shortcuts the T1-T2 bridging Cys-His covalent pathway. The role of the N δ1 H…OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV-vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1-T2 bridging chemical pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Mutagenesis

    International Nuclear Information System (INIS)

    Dubinin, N.P.

    1986-01-01

    Problems on radiation mutagenesis, in particular, data on general factors of genetic radiation effects, dependences of mutation frequencies on radiation dose and threshold in genetic radiation effects, problems of low doses, modification of genetic radiation effects, repauir of injuries of genetic material, photoreactivation, causing structure chromosomal mutations under radiation action, on relative genetic efficiency of different types of radiation are considered besides others

  15. Characterization of CYP154F1 from Thermobifida fusca YX and Extension of Its Substrate Spectrum by Site-Directed Mutagenesis.

    Science.gov (United States)

    Rühlmann, Ansgar; Groth, Georg; Urlacher, Vlada B

    2018-03-02

    Previous studies on cytochrome P450 monooxygenases (CYP) from family 154 reported their substrate promiscuity and high activity. Hence, herein, the uncharacterized family member CYP154F1 is described. Screening of more than 100 organic compounds revealed that CYP154F1 preferably accepts small linear molecules with a carbon chain length of 8-10 atoms. In contrast to thoroughly characterized CYP154E1, CYP154F1 has a much narrower substrate spectrum and lower activity. A structural alignment of homology models of CYP154F1 and CYP154E1 revealed few differences in the active sites of both family members. By gradual mutagenesis of the CYP154F1 active site towards those of CYP154E1, a key residue accounting for the different activities of both enzymes was identified at position 234. Substitution of T234 for large hydrophobic amino acids led to up to tenfold higher conversion rates of small substrates, such as geraniol. Replacement of T234 by small hydrophobic amino acids, valine or alanine, resulted in mutants with extended substrate spectra. These mutants are able to convert some of the larger substrates of CYP154E1, such as (E)-stilbene and (+)-nootkatone. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Study of signal transduction mechanism of angiotensin 2 receptor by means of site-directed mutagenesis; Bui totsuzen hen'iho wo mochiita anjiotenshin 2 reseputa no joho dentatsu kiko no kaimei

    Energy Technology Data Exchange (ETDEWEB)

    Yamano, Yoshiaki [Tottori University, Tottori (Japan). Faculty of Agriculture

    1998-12-16

    The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure. In order to clarify the signaling mechanism mediated by angiotensin 2 receptor, Gq-protein binding amino acid residues of this receptor were clarified by site-directed mutagenesis study. Amino acid residues in the carboxyl tail region were changed by alanines, individually. These mutated receptors were expressed stably in CHO cells, and GTP effect and second messenger molecules were determined, and three residues (Y 312, F313 and L 314) in this region were determined to be concerned for the binding of Gq protein. The other signaling systems, Gi, MAP kinase, JAK-STAT mediated, were reported to be concerned for this receptor. Novel drags for high blood pressure therapy would be explored by clarifying these signaling mechanisms. (author)

  17. Ligand-bound Structures and Site-directed Mutagenesis Identify the Acceptor and Secondary Binding Sites of Streptomyces coelicolor Maltosyltransferase GlgE*

    Science.gov (United States)

    Syson, Karl; Stevenson, Clare E. M.; Miah, Farzana; Barclay, J. Elaine; Tang, Minhong; Gorelik, Andrii; Rashid, Abdul M.; Lawson, David M.; Bornemann, Stephen

    2016-01-01

    GlgE is a maltosyltransferase involved in α-glucan biosynthesis in bacteria that has been genetically validated as a target for tuberculosis therapies. Crystals of the Mycobacterium tuberculosis enzyme diffract at low resolution so most structural studies have been with the very similar Streptomyces coelicolor GlgE isoform 1. Although the donor binding site for α-maltose 1-phosphate had been previously structurally defined, the acceptor site had not. Using mutagenesis, kinetics, and protein crystallography of the S. coelicolor enzyme, we have now identified the +1 to +6 subsites of the acceptor/product, which overlap with the known cyclodextrin binding site. The sugar residues in the acceptor subsites +1 to +5 are oriented such that they disfavor the binding of malto-oligosaccharides that bear branches at their 6-positions, consistent with the known acceptor chain specificity of GlgE. A secondary binding site remote from the catalytic center was identified that is distinct from one reported for the M. tuberculosis enzyme. This new site is capable of binding a branched α-glucan and is most likely involved in guiding acceptors toward the donor site because its disruption kinetically compromises the ability of GlgE to extend polymeric substrates. However, disruption of this site, which is conserved in the Streptomyces venezuelae GlgE enzyme, did not affect the growth of S. venezuelae or the structure of the polymeric product. The acceptor subsites +1 to +4 in the S. coelicolor enzyme are well conserved in the M. tuberculosis enzyme so their identification could help inform the design of inhibitors with therapeutic potential. PMID:27531751

  18. Assigning the EPR Fine Structure Parameters of the Mn(II) Centers in Bacillus subtilis Oxalate Decarboxylase by Site-Directed Mutagenesis and DFT/MM Calculations

    Science.gov (United States)

    2015-01-01

    Oxalate decarboxylase (OxDC) catalyzes the Mn-dependent conversion of the oxalate monoanion into CO2 and formate. EPR-based strategies for investigating the catalytic mechanism of decarboxylation are complicated by the difficulty of assigning the signals associated with the two Mn(II) centers located in the N- and C-terminal cupin domains of the enzyme. We now report a mutational strategy that has established the assignment of EPR fine structure parameters to each of these Mn(II) centers at pH 8.5. These experimental findings are also used to assess the performance of a multistep strategy for calculating the zero-field splitting parameters of protein-bound Mn(II) ions. Despite the known sensitivity of calculated D and E values to the computational approach, we demonstrate that good estimates of these parameters can be obtained using cluster models taken from carefully optimized DFT/MM structures. Overall, our results provide new insights into the strengths and limitations of theoretical methods for understanding electronic properties of protein-bound Mn(II) ions, thereby setting the stage for future EPR studies on the electronic properties of the Mn(II) centers in OxDC and site-specific variants. PMID:24444454

  19. Identification of functional sites in the catalytic subunit of cAMP-dependent protein kinase by differential modification and site-directed mutagenesis

    International Nuclear Information System (INIS)

    Buechler, J.A.

    1989-01-01

    In order to characterize substrate-induced conformational changes in the catalytic (C) subunit of cAMP-dependent protein kinase, the reactivity of the lysine residues towards [ 3 H]-acetic anhydride was determined in the absence of substrates, with MgATP bound to the enzyme, and when MgATP and an inhibitor peptide were present. A model for a portion of the ATP binding site in the C subunit was proposed based in part on the changes in lysine reactivity induced by MgATP binding to the C subunit. An intramolecular cross-link between a carboxyl group activated by dicyclohexylcarbodiimide (DCCD) and a lysine residue was circumvented by first modifying the lysine residues in the C subunit with acetic anhydride, and then labeling the modified C subunit with DCCD and [ 14 C]-glycine ethyl ester. Two carboxyl groups, Asp 184 and Glu 91, were labeled in the apoenzyme, but protected from modification in the presence of MgATP. The two residues accounting for the intramolecular cross-link mediated by DCCD were identified by first labeling the apoenzyme with DCCD, followed by modification of the lysine residues with [ 3 H]-acetic anhydride. The two residues involved in the cross-link, Asp 184 and Lys 72, are both invariant amino acids in the protein kinase family, and a potential orientation of the active site was proposed. The C subunit was modified with a water soluble carbodiimide, 1-ethyl-3-(3-dimethyl-amino-propyl) carbodiimide (EDC) and [ 14 C]glycine ethyl ester in order to identify carboxyl groups that may interact with the basic residues of the protein substrates. Either MgATP or peptide inhibitor alone did not protect the C subunit from inhibition, but together they blocked the inactivation by EDC

  20. Site-directed mutagenesis of putative substrate recognition sites in cytochrome P450 2B11: importance of amino acid residues 114, 290, and 363 for substrate specificity.

    Science.gov (United States)

    Hasler, J A; Harlow, G R; Szklarz, G D; John, G H; Kedzie, K M; Burnett, V L; He, Y A; Kaminsky, L S; Halpert, J R

    1994-08-01

    Eleven amino acid residues unique to dog cytochrome P450 (P450) 2B11, compared with rat 2B1 and 2B2, rabbit 2B4 and 2B5, and mouse 2B10, in the putative substrate recognition sites [J. Biol. Chem. 267:83-90 (1992)] were mutated to the residues found in 2B1 or 2B5. The mutants were expressed initially in COS cells and screened for activity toward androstenedione and 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB). P450 2B11 mutants V107I, M199L-N200E-V204R, V234I, A292L, Q473R, and I475S showed no differences from wild-type P450 2B11 in metabolite profiles with either substrate. Mutants V114I, D290I, and L363V exhibited altered androstenedione metabolite profiles and were expressed in Escherichia coli for further study with androstenedione, testosterone, 7-ethoxycoumarin, (R)- and (S)-warfarin, and 245-HCB. With V114I, hydroxylation of steroids and warfarin and 2-hydroxylation of 245-HCB were decreased, whereas 7-ethoxycoumarin O-dealkylation and 3-hydroxylation of 245-HCB were unaltered. For D290I, activities toward all substrates were decreased, except for 16 beta-hydroxylation of testosterone. The activity of L363V was increased 5-6-fold for 16 alpha-hydroxylation of androstenedione and testosterone but was decreased to 40-50% of wild-type activity with 7-ethoxycoumarin and warfarin and to 6-8% of control for 2-hydroxylation of 245-HCB. Alignment of P450 2B11 with P450 101 and super-imposition of the 11 mutated 2B11 residues on a P450 101 three-dimensional model suggest that only residues 114, 290, and 363 represent substrate contact residues, in excellent agreement with the experimental results. The data indicate the importance of the three residues 114, 290, and 363 in substrate specificity and regio- and stereoselectivity of P450 2B11 and also demonstrate that the effects of the mutations vary considerably with different substrates.

  1. Role of the conserved amino acids of the 'SDN' loop (Ser130, Asp131 and Asn132) in a class A beta-lactamase studied by site-directed mutagenesis.

    Science.gov (United States)

    Jacob, F; Joris, B; Lepage, S; Dusart, J; Frère, J M

    1990-10-15

    Ser130, Asp131 and Asn132 ('SDN') are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.

  2. ENU mutagenesis to generate genetically modified rat models.

    Science.gov (United States)

    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.

  3. Structure-function relationships in the Na,K-ATPase α subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

    International Nuclear Information System (INIS)

    Price, E.M.; Lingrel, J.B.

    1988-01-01

    Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the α1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat α1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase α subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep α1 subunit was changed to that of the rat. When expressed in HeLa cells, this mutated sheep α1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep α1 cDNA containing only two amino acid substitutions. The resistant cells, whether transfected with the rat α1 cDNA, the rat/sheep chimera, or the mutant sheep α1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, 86 Rb + uptake, and Na,K-ATPase activity. These results demonstrate that the presence of arginine and aspartic acid on the amino end and carboxyl end, respectively, of the H1-H2 extracellular domain of the Na,K-ATPase α subunit together is responsible for the ouabain-resistant character of the rat enzyme and the corresponding residues in the sheep α1 subunit (glutamine and asparagine) are somehow involved in ouabain binding

  4. Structure-function relationships in the Na,K-ATPase. cap alpha. subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Price, E.M.; Lingrel, J.B.

    1988-11-01

    Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the ..cap alpha..1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat ..cap alpha..1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase ..cap alpha.. subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep ..cap alpha..1 subunit was changed to that of the rat. When expressed in HeLa cells, this mutated sheep ..cap alpha..1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep ..cap alpha..1 cDNA containing only two amino acid substitutions. The resistant cells, whether transfected with the rat ..cap alpha..1 cDNA, the rat/sheep chimera, or the mutant sheep ..cap alpha..1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, /sup 86/Rb/sup +/ uptake, and Na,K-ATPase activity. These results demonstrate that the presence of arginine and aspartic acid on the amino end and carboxyl end, respectively, of the H1-H2 extracellular domain of the Na,K-ATPase ..cap alpha.. subunit together is responsible for the ouabain-resistant character of the rat enzyme and the corresponding residues in the sheep ..cap alpha..1 subunit (glutamine and asparagine) are somehow involved in ouabain binding.

  5. Stationary-state mutagenesis in Escherichia coli: a model

    Indian Academy of Sciences (India)

    Here we propose a more detailed version of this model that also takes into account the observed genetic requirements of stationary-state mutagenesis. Briefly, G:T/U mismatches produced at methylatable cytosines are preferentially repaired in nondividing cells by the very short patch mismatch repair (VSPMR) mechanism ...

  6. Dopamine D1 receptor-agonist interactions: A mutagenesis and homology modeling study.

    Science.gov (United States)

    Mente, Scot; Guilmette, Edward; Salafia, Michelle; Gray, David

    2015-01-01

    The dopamine D1 receptor is a G protein-coupled receptor that regulates intracellular signaling via agonist activation. Although the number of solved GPCR X-ray structures has been steadily increasing, still no structure of the D1 receptor exists. We have used site-directed mutagenesis of 12 orthosteric vicinity residues of possible importance to G protein-coupled activation to examine the function of prototypical orthosteric D1 agonists and partial agonists. We find that residues from four different regions of the D1 receptor make significant contributions to agonist function. All compounds studied, which are catechol-amines, are found to interact with the previously identified residues: the conserved D103(3.32), as well as the trans-membrane V serine residues. Additional key interactions are found for trans-membrane VI residues F288(6.51), F289(6.52) and N292(6.55), as well as the extra-cellular loop residue L190(ECL2). Molecular dynamics simulations of a D1 homology model have been used to help put the ligand-residue interactions into context. Finally, we considered the rescaling of fold-shift data as a method to account for the change in the size of the mutated side-chain and found that this rescaling helps to relate the calculated ligand-residue energies with observed experimental fold-shifts. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Functional Differentiation of Antiporter-Like Polypeptides in Complex I; a Site-Directed Mutagenesis Study of Residues Conserved in MrpA and NuoL but Not in MrpD, NuoM, and NuoN.

    Directory of Open Access Journals (Sweden)

    Eva Sperling

    Full Text Available It has long been known that the three largest subunits in the membrane domain (NuoL, NuoM and NuoN of complex I are homologous to each other, as well as to two subunits (MrpA and MrpD from a Na+/H+ antiporter, Mrp. MrpA and NuoL are more similar to each other and the same is true for MrpD and NuoN. This suggests a functional differentiation which was proven experimentally in a deletion strain model system, where NuoL could restore the loss of MrpA, but not that of MrpD and vice versa. The simplest explanation for these observations was that the MrpA and MrpD proteins are not antiporters, but rather single subunit ion channels that together form an antiporter. In this work our focus was on a set of amino acid residues in helix VIII, which are only conserved in NuoL and MrpA (but not in any of the other antiporter-like subunits. and to compare their effect on the function of these two proteins. By combining complementation studies in B. subtilis and 23Na-NMR, response of mutants to high sodium levels were tested. All of the mutants were able to cope with high salt levels; however, all but one mutation (M258I/M225I showed differences in the efficiency of cell growth and sodium efflux. Our findings showed that, although very similar in sequence, NuoL and MrpA seem to differ on the functional level. Nonetheless the studied mutations gave rise to interesting phenotypes which are of interest in complex I research.

  8. Efficient multi-site-directed mutagenesis directly from genomic ...

    Indian Academy of Sciences (India)

    2012-09-29

    Sep 29, 2012 ... ly by using one universal and one mutagenic primer or two mutagenic primer pairs. The two or more intermediate prod- ucts with complementary ends form a new template DNA by allowing the 3′ overlap of each strand to serve as primer for .... bined products were transformed to Escherichia coli (DH5α).

  9. Molecular cloning, homology modeling and site-directed mutagenesis of vanadium-dependent bromoperoxidase (GcVBPO1) from Gracilaria changii (Rhodophyta).

    Science.gov (United States)

    Baharum, H; Chu, W-C; Teo, S-S; Ng, K-Y; Rahim, R Abdul; Ho, C-L

    2013-08-01

    Vanadium-dependent haloperoxidases belong to a class of vanadium enzymes that may have potential industrial and pharmaceutical applications due to their high stability. In this study, the 5'-flanking genomic sequence and complete reading frame encoding vanadium-dependent bromoperoxidase (GcVBPO1) was cloned from the red seaweed, Fracilaria changii, and the recombinant protein was biochemically characterized. The deduced amino acid sequence of GcVBPO1 is 1818 nucleotides in length, sharing 49% identity with the vanadium-dependent bromoperoxidases from Corralina officinalis and Cor. pilulifera, respectively. The amino acid residues associated with the binding site of vanadate cofactor were found to be conserved. The Km value of recombinant GcVBPO1 for Br(-) was 4.69 mM, while its Vmax was 10.61 μkat mg(-1) at pH 7. Substitution of Arg(379) with His(379) in the recombinant protein caused a lower affinity for Br(-), while substitution of Arg(379) with Phe(379) not only increased its affinity for Br(-) but also enabled the mutant enzyme to oxidize Cl(-). The mutant Arg(379)Phe was also found to have a lower affinity for I(-), as compared to the wild-type GcVBPO1 and mutant Arg(379)His. In addition, the Arg(379)Phe mutant has a slightly higher affinity for H2O2 compared to the wild-type GcVBPO1. Multiple cis-acting regulatory elements associated with light response, hormone signaling, and meristem expression were detected at the 5'-flanking genomic sequence of GcVBPO1. The transcript abundance of GcVBPO1 was relatively higher in seaweed samples treated with 50 parts per thousand (ppt) artificial seawater (ASW) compared to those treated in 10 and 30 ppt ASW, in support of its role in the abiotic stress response of seaweed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Katz model prediction of Caenorhabditis elegans mutagenesis on STS-42

    Science.gov (United States)

    Cucinotta, Francis A.; Wilson, John W.; Katz, Robert; Badhwar, Gautam D.

    1992-01-01

    Response parameters that describe the production of recessive lethal mutations in C. elegans from ionizing radiation are obtained with the Katz track structure model. The authors used models of the space radiation environment and radiation transport to predict and discuss mutation rates for C. elegans on the IML-1 experiment aboard STS-42.

  11. UV-induced mutagenesis in Escherichia coli SOS response: a quantitative model.

    Directory of Open Access Journals (Sweden)

    Sandeep Krishna

    2007-03-01

    Full Text Available Escherichia coli bacteria respond to DNA damage by a highly orchestrated series of events known as the SOS response, regulated by transcription factors, protein-protein binding, and active protein degradation. We present a dynamical model of the UV-induced SOS response, incorporating mutagenesis by the error-prone polymerase, Pol V. In our model, mutagenesis depends on a combination of two key processes: damage counting by the replication forks and a long-term memory associated with the accumulation of UmuD'. Together, these provide a tight regulation of mutagenesis, resulting, we show, in a "digital" turn-on and turn-off of Pol V. Our model provides a compact view of the topology and design of the SOS network, pinpointing the specific functional role of each of the regulatory processes. In particular, we suggest that the recently observed second peak in the activity of promoters in the SOS regulon (Friedman et al., 2005, PLoS Biology 3(7: e238 is the result of positive feedback from Pol V to RecA filaments.

  12. ENU mutagenesis to generate genetically modified rat models

    NARCIS (Netherlands)

    van Boxtel, R.; Gould, M.; Cuppen, E.; Smits, B.M.

    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

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

    Directory of Open Access Journals (Sweden)

    Mitsuru eFurusawa

    2014-12-01

    Full Text Available In animals including humans, mutation rates per generation will exceed a perceived threshold, which should result in an excessive genetic load. Despite this, they have survived without extinction. This is a perplexing problem for 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 there is a high enough fidelity difference in replication between the lagging and leading strands. 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-prepared 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.

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

    Science.gov (United States)

    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

  15. Comparative Analysis of Context-Dependent Mutagenesis Using Human and Mouse Models

    Directory of Open Access Journals (Sweden)

    Sofya A. Medvedeva

    2013-01-01

    Full Text Available Substitution rates strongly depend on their nucleotide context. One of the most studied examples is the excess of C > T mutations in the CG context in various groups of organisms, including vertebrates. Studies on the molecular mechanisms underlying this mutation regularity have provided insights into evolution, mutagenesis, and cancer development. Recently several other hypermutable motifs were identified in the human genome. There is an increased frequency of T > C mutations in the second position of the words ATTG and ATAG and an increased frequency of A > C mutations in the first position of the word ACAA. For a better understanding of evolution, it is of interest whether these mutation regularities are human specific or present in other vertebrates, as their presence might affect the validity of currently used substitution models and molecular clocks. A comprehensive analysis of mutagenesis in 4 bp mutation contexts requires a vast amount of mutation data. Such data may be derived from the comparisons of individual genomes or from single nucleotide polymorphism (SNP databases. Using this approach, we performed a systematical comparison of mutation regularities within 2–4 bp contexts in Mus musculus and Homo sapiens and uncovered that even closely related organisms may have notable differences in context-dependent mutation regularities.

  16. Directed evolution and targeted mutagenesis to murinize listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model

    Directory of Open Access Journals (Sweden)

    Hill Colin

    2010-12-01

    Full Text Available Abstract Background Internalin A (InlA is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells. Results We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26, multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlAm murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlAm*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlAm* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlAm strain. Conclusions We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced

  17. Directed evolution and targeted mutagenesis to murinize Listeria monocytogenes Internalin A for enhanced infectivity in the murine oral infection model

    LENUS (Irish Health Repository)

    Monk, Ian R

    2010-12-13

    Abstract Background Internalin A (InlA) is a critical virulence factor which mediates the initiation of Listeria monocytogenes infection by the oral route in permissive hosts. The interaction of InlA with the host cell ligand E-cadherin efficiently stimulates L. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells. Results We have created a surface display library of randomly mutated InlA in a non-invasive heterologous host Lactococcus lactis in order to create and screen novel variants of this invasion factor. After sequential passage through a murine cell line (CT-26), multiple clones with enhanced invasion characteristics were identified. Competitive index experiments were conducted in mice using selected mutations introduced into L. monocytogenes EGD-e background. A novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. As a control a previously described EGD-InlAm murinized strain was also re-created as part of this study with minor modifications and designated EGD-e InlA m*. The strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates Listeria-optimized codons encoding the altered amino acids. L. monocytogenes EGD-e InlA m* yielded consistently higher level murine infections by the oral route when compared to EGD-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the EGD-InlAm strain. Conclusions We have used both site-directed mutagenesis and directed evolution to create variants of InlA which may inform future structure-function analyses of this protein. During the course of the study we engineered a murinized strain of L. monocytogenes EGD-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced entry into human

  18. Creation of miniature pig model of human Waardenburg syndrome type 2A by ENU mutagenesis.

    Science.gov (United States)

    Hai, Tang; Guo, Weiwei; Yao, Jing; Cao, Chunwei; Luo, Ailing; Qi, Meng; Wang, Xianlong; Wang, Xiao; Huang, Jiaojiao; Zhang, Ying; Zhang, Hongyong; Wang, Dayu; Shang, Haitao; Hong, Qianlong; Zhang, Rui; Jia, Qitao; Zheng, Qiantao; Qin, Guosong; Li, Yongshun; Zhang, Tao; Jin, Weiwu; Chen, Zheng-Yi; Wang, Hongmei; Zhou, Qi; Meng, Anming; Wei, Hong; Yang, Shiming; Zhao, Jianguo

    2017-11-01

    Human Waardenburg syndrome 2A (WS2A) is a dominant hearing loss (HL) syndrome caused by mutations in the microphthalmia-associated transcription factor (MITF) gene. In mouse models with MITF mutations, WS2A is transmitted in a recessive pattern, which limits the study of hearing loss (HL) pathology. In the current study, we performed ENU (ethylnitrosourea) mutagenesis that resulted in substituting a conserved lysine with a serine (p. L247S) in the DNA-binding domain of the MITF gene to generate a novel miniature pig model of WS2A. The heterozygous mutant pig (MITF +/L247S ) exhibits a dominant form of profound HL and hypopigmentation in skin, hair, and iris, accompanied by degeneration of stria vascularis (SV), fused hair cells, and the absence of endocochlear potential, which indicate the pathology of human WS2A. Besides hypopigmentation and bilateral HL, the homozygous mutant pig (MITF L247S/L247S ) and CRISPR/Cas9-mediated MITF bi-allelic knockout pigs both exhibited anophthalmia. Three WS2 patients carrying MITF mutations adjacent to the corresponding region were also identified. The pig models resemble the clinical symptom and molecular pathology of human WS2A patients perfectly, which will provide new clues for better understanding the etiology and development of novel treatment strategies for human HL.

  19. Spontaneous inflammatory pain model from a mouse line with N-ethyl-N-nitrosourea mutagenesis

    Directory of Open Access Journals (Sweden)

    Chen Tsung-Chieh

    2012-05-01

    in pstpip2 causes autoinflammatory disease in an N-ethyl-N-nitrosourea mutagenesis mouse model. Thus, our pstpip2 mutant mice provide a new model for investigating the potential mechanisms of inflammatory pain.

  20. Optimization of Combinatorial Mutagenesis

    Science.gov (United States)

    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. Correlation of binding-loop internal dynamics with stability and function in potato I inhibitor family: relative contributions of Arg(50) and Arg(52) in Cucurbita maxima trypsin inhibitor-V as studied by site-directed mutagenesis and NMR spectroscopy.

    Science.gov (United States)

    Cai, Mengli; Gong, Yu-Xi; Wen, Lisa; Krishnamoorthi, Ramaswamy

    2002-07-30

    The side chains of Arg(50) and Arg(52) at positions P(6)' and P(8)', respectively, anchor the binding loop to the protein scaffold by means of hydrogen bonds in Cucurbita maxima trypsin inhibitor-V (CMTI-V), a potato I family member. Here, we have investigated the relative contributions of Arg(50) and Arg(52) to the binding-loop flexibility and stability by determining changes in structure, dynamics, and proteolytic stability as a consequence of individually mutating them into an alanine. We have compared chemical shift assignments of main-chain hydrogens and nitrogens, and (1)H-(1)H interresidue nuclear Overhauser effects (NOEs) for the two mutants with those of the wild-type protein. We have also measured NMR longitudinal and transverse relaxation rates and (15)N-(1)H NOE enhancements for all backbone and side-chain NH groups and calculated the model-free parameters for R50A-rCMTI-V and R52A-rCMTI-V. The three-dimensional structures and backbone dynamics of the protein scaffold region remain very similar for both mutants, relative to the wild-type protein. The flexibility of the binding loop is increased in both R50A- and R52A-rCMTI-V. In R52A-rCMTI-V, the mean generalized order parameter () of the P(6)-P(1) residues of the binding loop (39-44) decreases to 0.68 +/- 0.02 from 0.76 +/- 0.04 observed for the wild-type protein. However, in R50A-rCMTI-V, the flexibility of the whole binding loop increases, especially that of the P(1)'-P(3)' residues (45-47), whose value drops dramatically to 0.35 +/- 0.03 from 0.68 +/- 0.03 determined for rCMTI-V. More strikingly, S(2) values of side-chain N epsilon Hs reveal that, in the R50A mutant, removal of the R50 hydrogen bond results in the loss of the R52 hydrogen bond too, whereas in R52A, the R50 hydrogen bond remains unaffected. Kinetic data on trypsin-catalyzed hydrolysis of the reactive-site peptide bond (P(1)-P(1)') suggest that the activation free energy barrier of the reaction at 25 degrees C is reduced by 2.1 kcal

  2. Characterization and site-directed mutagenesis of Wzb, an O-phosphatase from Lactobacillus rhamnosus

    Directory of Open Access Journals (Sweden)

    Gilbert Christophe

    2008-04-01

    Full Text Available Abstract Background Reversible phosphorylation events within a polymerisation complex have been proposed to modulate capsular polysaccharide synthesis in Streptococcus pneumoniae. Similar phosphatase and kinase genes are present in the exopolysaccharide (EPS biosynthesis loci of numerous lactic acid bacteria genomes. Results The protein sequence deduced from the wzb gene in Lactobacillus rhamnosus ATCC 9595 reveals four motifs of the polymerase and histidinol phosphatase (PHP superfamily of prokaryotic O-phosphatases. Native and modified His-tag fusion Wzb proteins were purified from Escherichia coli cultures. Extracts showed phosphatase activity towards tyrosine-containing peptides. The purified fusion protein Wzb was active on p-nitrophenyl-phosphate (pNPP, with an optimal activity in presence of bovine serum albumin (BSA 1% at pH 7.3 and a temperature of 75°C. At 50°C, residual activity decreased to 10 %. Copper ions were essential for phosphatase activity, which was significantly increased by addition of cobalt. Mutated fusion Wzb proteins exhibited reduced phosphatase activity on p-nitrophenyl-phosphate. However, one variant (C6S showed close to 20% increase in phosphatase activity. Conclusion These characteristics reveal significant differences with the manganese-dependent CpsB protein tyrosine phosphatase described for Streptococcus pneumoniae as well as with the polysaccharide-related phosphatases of Gram negative bacteria.

  3. Site directed mutagenesis of Drosophila flightin disrupts phosphorylation and impairs flight muscle structure and mechanics.

    Science.gov (United States)

    Barton, Byron; Ayer, Gretchen; Maughan, David W; Vigoreaux, Jim O

    2007-01-01

    Flightin is a myosin rod binding protein that in Drosophila melanogaster is expressed exclusively in the asynchronous indirect flight muscles (IFM). Hyperphosphorylation of flightin coincides with the completion of myofibril assembly and precedes the emergence of flight competency in young adults. To investigate the role of flightin phosphorylation in vivo we generated three flightin null (fln(0)) Drosophila strains that express a mutant flightin transgene with two (Thr158, Ser 162), three (Ser139, Ser141, Ser145) or all five potential phosphorylation sites mutated to alanines. These amino acid substitutions result in lower than normal levels of flightin accumulation and transgenic strains that are unable to beat their wings. On two dimensional gels of IFM proteins, the transgenic strain with five mutant sites (fln(5STA)) is devoid of all phosphovariants, the transgenic strain with two mutant sites (fln(2TSA)) expresses only the two least acidic of the nine phosphovariants, and the transgenic strain with three mutant sites (fln(3SA)) expresses all nine phosphovariants, as the wild-type strain. These results suggest that phosphorylation of Thr158 and/or Ser162 is necessary for subsequent phosphorylation of other sites. All three transgenic strains show normal, albeit long, IFM sarcomeres in newly eclosed adults. In contrast, sarcomeres in fully mature fln(5STA) and fln(2TSA) adults show extensive breakdown while those in fln(3SA) are not as disordered. The fiber hypercontraction phenotype that characterizes fln(0) is fully evident in fln(5STA) and fln(2TSA) but partially rescued in fln(3SA). Mechanics on skinned fibers from newly eclosed flies show alterations in viscous modulus for fln(5STA) and fln(2TSA) that result in a significant reduction in oscillatory power output. Expression of fln(5STA) and fln(2TSA), but not fln(3SA), in a wild-type (fln(+)/fln(+)) background resulted in a dominant negative effect manifested as flight impairments and hypercontracted IFM fibers. Our studies indicate that Thr158 and/or Ser162 are (is) indispensable for flightin function and suggest that phosphorylation of one or both residues fulfills an essential role in IFM structural stability and mechanics.

  4. Antifreeze activity enhancement by site directed mutagenesis on an antifreeze protein from the beetle Rhagium mordax

    DEFF Research Database (Denmark)

    Friis, Dennis Steven; Kristiansen, Erlend; von Solms, Nicolas

    2014-01-01

    The ice binding motifs of insect antifreeze proteins (AFPs) mainly consist of repetitive TxT motifs aligned on a flat face of the protein. However, these motifs often contain non-threonines that disrupt the TxT pattern. We substituted two such disruptive amino acids located in the ice binding face...... of an AFP from Rhagium mordax with threonine. Furthermore, a mutant with an extra ice facing TxT motif was constructed. These mutants showed enhanced antifreeze activity compared to the wild type at low concentrations. However, extrapolating the data indicates that the wild type will become the most active...

  5. Molecular determinants of ligand binding modes in the histamine H(4) receptor: linking ligand-based three-dimensional quantitative structure-activity relationship (3D-QSAR) models to in silico guided receptor mutagenesis studies.

    Science.gov (United States)

    Istyastono, Enade P; Nijmeijer, Saskia; Lim, Herman D; van de Stolpe, Andrea; Roumen, Luc; Kooistra, Albert J; Vischer, Henry F; de Esch, Iwan J P; Leurs, Rob; de Graaf, Chris

    2011-12-08

    The histamine H(4) receptor (H(4)R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H(3) receptor (H(3)R), two acidic residues in the H(4)R binding pocket, D(3.32) and E(5.46), act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H(4)R ligands. Given the symmetric distribution of these complementary pharmacophore features in H(4)R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H(4)R-ligand binding modes by combining (3D) quantitative structure-activity relationship (QSAR), protein homology modeling, molecular dynamics simulations, and site-directed mutagenesis studies. We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea) derivatives to investigate H(4)R-ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H(4)R in two distinct binding modes, while the addition of a cyclohexyl group to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H(4)R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H(4)R and can be used as a general approach to elucidate the structure of protein-ligand complexes.

  6. A screening system to identify transcription factors that induce binding site-directed DNA demethylation.

    Science.gov (United States)

    Suzuki, Takahiro; Maeda, Shiori; Furuhata, Erina; Shimizu, Yuri; Nishimura, Hajime; Kishima, Mami; Suzuki, Harukazu

    2017-12-08

    DNA methylation is a fundamental epigenetic modification that is involved in many biological systems such as differentiation and disease. We and others recently showed that some transcription factors (TFs) are involved in the site-specific determination of DNA demethylation in a binding site-directed manner, although the reports of such TFs are limited. Here, we develop a screening system to identify TFs that induce binding site-directed DNA methylation changes. The system involves the ectopic expression of target TFs in model cells followed by DNA methylome analysis and overrepresentation analysis of the corresponding TF binding motif at differentially methylated regions. It successfully identified binding site-directed demethylation of SPI1, which is known to promote DNA demethylation in a binding site-directed manner. We extended our screening system to 15 master TFs involved in cellular differentiation and identified eight novel binding site-directed DNA demethylation-inducing TFs (RUNX3, GATA2, CEBPB, MAFB, NR4A2, MYOD1, CEBPA, and TBX5). Gene ontology and tissue enrichment analysis revealed that these TFs demethylate genomic regions associated with corresponding biological roles. We also describe the characteristics of binding site-directed DNA demethylation induced by these TFs, including the targeting of highly methylated CpGs, local DNA demethylation, and the overlap of demethylated regions between TFs of the same family. Our results show the usefulness of the developed screening system for the identification of TFs that induce DNA demethylation in a site-directed manner.

  7. Biochemical and structural analysis of a site directed mutant of manganese dependent aminopeptidase P from Streptomyces lavendulae

    Directory of Open Access Journals (Sweden)

    ARYA NANDAN

    2015-08-01

    Full Text Available Aminopeptidase P (APP removes N-terminal amino acids from peptides and proteins when the penultimate residue is proline. To understand the structure-function relationships of aminopeptidase P of Streptomyces lavendulae, a conserved arginine residue was replaced with lysine (R453K by site-directed mutagenesis. The overexpressed wild and mutant enzymes were of nearly 60 kDa and purified by nickel affinity chromatography. Kinetic analysis of R453K variant using Gly-Pro-pNA as the substrate revealed an increase in Km with a decrease in Vmax, leading to overall decrease in the catalytic efficiency, indicating that the guanidinium group of arginine plays an important role in substrate binding in APP. We constructed three dimensional models for the catalytic domains of wild and mutant enzyme and it revealed an interaction in R453 of native enzyme through hydrogen bonding with the adjacent residues making a substrate binding cavity whereas K453 did not participate in any hydrogen bonding. Hence, R453 in APP of S. lavenduale must be playing a critical role in the hydrolysis of the substrate.

  8. Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red®/ET® Recombination

    Directory of Open Access Journals (Sweden)

    Jung Kirsten

    2008-04-01

    Full Text Available Abstract Background Introducing point mutations into bacterial chromosomes is important for further progress in studies relying on functional genomics, systems- and synthetic biology, and for metabolic engineering. For many investigations, chromosomal systems are required rather than artificial plasmid based systems. Results Here we describe the introduction of a single point mutation into the Escherichia coli chromosome by site-directed mutagenesis without leaving any selection marker. We used Red®/ET® Recombination in combination with rpsL counter-selection to introduce a single point mutation into the E. coli MG1655 genome, one of the widely used bacterial model strains in systems biology. The method we present is rapid and highly efficient. Since single-stranded synthetic oligonucleotides can be used for recombination, any chromosomal modification can be designed. Conclusion Chromosomal modifications performed by rpsL counter-selection may also be used for other bacteria that contain an rpsL homologue, since Red®/ET® Recombination has been applied to several enteric bacteria before.

  9. Combining modelling and mutagenesis studies of synaptic vesicle protein 2A to identify a series of residues involved in racetam binding.

    Science.gov (United States)

    Shi, Jiye; Anderson, Dina; Lynch, Berkley A; Castaigne, Jean-Gabriel; Foerch, Patrik; Lebon, Florence

    2011-10-01

    LEV (levetiracetam), an antiepileptic drug which possesses a unique profile in animal models of seizure and epilepsy, has as its unique binding site in brain, SV2A (synaptic vesicle protein 2A). Previous studies have used a chimaeric and site-specific mutagenesis approach to identify three residues in the putative tenth transmembrane helix of SV2A that, when mutated, alter binding of LEV and related racetam derivatives to SV2A. In the present paper, we report a combined modelling and mutagenesis study that successfully identifies another 11 residues in SV2A that appear to be involved in ligand binding. Sequence analysis and modelling of SV2A suggested residues equivalent to critical functional residues of other MFS (major facilitator superfamily) transporters. Alanine scanning of these and other SV2A residues resulted in the identification of residues affecting racetam binding, including Ile273 which differentiated between racetam analogues, when mutated to alanine. Integrating mutagenesis results with docking analysis led to the construction of a mutant in which six SV2A residues were replaced with corresponding SV2B residues. This mutant showed racetam ligand-binding affinity intermediate to the affinities observed for SV2A and SV2B.

  10. Preliminary Work in Obtaining Site-Directed Mutants of Hen Egg White Lysozyme

    Science.gov (United States)

    Holmes, Leonard D.

    1996-01-01

    tetramer octamer higher order. It is believed that multimer aggregation of lysozyme occurs by interaction at specific binding sites on the surface of the protein crystals. If the presence of discrete binding sites and the aggregation hypothesis is true, then it follows that the alteration of the binding site(s) should have significant effect on the measurements obtained during growth experiments. Site-directed mutagenesis allows the specific alteration of proteins by replacement, deletion or addition of specific amino acid residues. This report outlines the approach for this strategy and the progress made thus far toward that end.

  11. Improved efficacy of soluble human receptor activator of nuclear factor kappa B (RANK) fusion protein by site-directed mutagenesis.

    Science.gov (United States)

    Son, Young Jun; Han, Jihye; Lee, Jae Yeon; Kim, HaHyung; Chun, Taehoon

    2015-06-01

    Soluble human receptor activator of nuclear factor kappa B fusion immunoglobulin (hRANK-Ig) has been considered as one of the therapeutic agents to treat osteoporosis or diseases associated with bone destruction by blocking the interaction between RANK and the receptor activator of nuclear factor kappa B ligand (RANKL). However, no scientific record showing critical amino acid residues within the structural interface between the human RANKL and RANK complex is yet available. In this study, we produced several mutants of hRANK-Ig by replacement of amino acid residue(s) and tested whether the mutants had increased binding affinity to human RANKL. Based on the results from flow cytometry and surface plasmon resonance analyses, the replacement of E(125) with D(125), or E(125) and C(127) with D(125) and F(127) within loop 3 of cysteine-rich domain 3 of hRANK-Ig increases binding affinity to human RANKL over the wild-type hRANK-Ig. This result may provide the first example of improvement in the efficacy of hRANK-Ig by protein engineering and may give additional information to understand a more defined structural interface between hRANK and RANKL.

  12. Increasing the transglycosylation activity of alpha-galactosidase from Bifidobacterium adolescentis DSM 20083 by site-directed mutagenesis

    NARCIS (Netherlands)

    Hinz, S.W.A.; Doeswijk-Voragen, C.H.L.; Schipperus, R.; Broek, van den L.A.M.; Vincken, J.P.; Voragen, A.G.J.

    2006-01-01

    The ¿-galactosidase (AGA) from Bifidobacterium adolescentis DSM 20083 has a high transglycosylation activity. The optimal conditions for this activity are pH 8, and 37°C. At high melibiose concentration (600 mM), approximately 64% of the enzyme-substrate encounters resulted in transglycosylation.

  13. Site-directed mutagenesis, kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp YM55-1

    NARCIS (Netherlands)

    Veetil, Vinod Puthan; Raj, Hans; Quax, Wim J.; Janssen, Dick B.; Poelarends, Gerrit J.

    Aspartate ammonia lyases (also referred to as aspartases) catalyze the reversible deamination of l-aspartate to yield fumarate and ammonia. In the proposed mechanism for these enzymes, an active site base abstracts a proton from C3 of l-aspartate to form an enzyme-stabilized enediolate intermediate.

  14. Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis.

    Science.gov (United States)

    Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2014-01-01

    We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170 kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50 kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl2, and HgCl2 functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl2 was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.

  15. Site-Directed Mutagenesis of Myoglobin for Studies of Their Interaction with Iron(III by Multi-Spectroscopic Techniques

    Directory of Open Access Journals (Sweden)

    Qian Tang

    2013-01-01

    Full Text Available In order to investigate how the amino acids on the surface of myoglobin molecule influence myoglobin's structure and function, a variety of spectroscopy techniques were applied in the study of the interaction between Fe(III and myoglobin (wild type and its mutants, D44K, D60K, and K56D. The results demonstrate that Fe(III can quench the fluorescence of wild type and mutants of myoglobin, and the quenching mechanisms are static quenching. It is found that the binding distance between Fe(III and myoglobin mutants gets smaller, the binding capacity increases by the values of binding constant and the bimolecular quenching constant as well as the binding distance. Those data also indicate that the metal ion Fe(III can interact strongly with myoglobin mutants. The three-dimensional conformation change after surface amino acids are replaced is detected by the UV absorption spectroscopy and fluorescence spectroscopy, which make mutants become more dynamic and change its function and interaction with Fe(III strongly.

  16. Thermostability enhancement of an endo-1,4-β-galactanase from Talaromyces stipitatus by site-directed mutagenesis

    DEFF Research Database (Denmark)

    Larsen, Dorte Møller; Nyffenegger, Christian; Swiniarska, Malgorzata Maria

    2015-01-01

    Enzymatic conversion of pectinaceous biomasses such as potato and sugar beet pulp at high temperatures is advantageous as it gives rise to lower substrate viscosity, easier mixing, and increased substrate solubility and lowers the risk of contamination. Such high-temperature processing requires d...

  17. Exploring residues crucial for nitrilase function by site directed mutagenesis to gain better insight into sequence-function relationships.

    Science.gov (United States)

    Kaushik, Shubhangi; Mohan, Utpal; Banerjee, Uc

    2012-01-01

    Nitrilases represent a very important class of enzymes having an array of applications. In the present scenario, where the indepth information about nitrilases is limited, the present work is an attempt to shed light on the residues crucial for the nitrilase activity. The nitrilase sequences demonstrating varying degree of identity with P. putida nitrilase were explored. A stretch of residues, fairly conserved throughout the range of higher (96%) to lower (27%) sequence identity among different nitrilases was selected and investigated for the possible functional role in nitrilase enzyme system. Subsequently, the alanine substitution mutants (T48A, W49A, L50A, P51A, G52A, Y53A and P54A) were generated. Substitution of the rationally selected conserved residues altered the substrate recognition ability, catalysis and affected the substrate specificity but had very little impact on enantioselectivity and pattern of nitrile hydrolysis.

  18. Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121

    NARCIS (Netherlands)

    Ozimek, L.K.; Hijum, S.A.F.T. van; Koningsveld, G.A. van; Maarel, M.J.E.C. van der; Geel-Schutten, G.H. van; Dijkhuizen, L.

    2004-01-01

    Bacterial fructosyltransferases (FTFs) are retaining-type glycosidases that belong to family 68 of glycoside hydrolases. Recently, the high-resolution 3D structure of the Bacillus subtilis levansucrase has been solved [Meng, G. and Futterer, K., Nat. Struct. Biol. 10 (2003) 935-941]. Based on this

  19. Affinity enhancement of nanobody binding to EGFR: in silico site-directed mutagenesis and molecular dynamics simulation approaches.

    Science.gov (United States)

    Farasat, Alireza; Rahbarizadeh, Fatemeh; Hosseinzadeh, Ghader; Sajjadi, Sharareh; Kamali, Mehdi; Keihan, Amir Homayoun

    2017-06-01

    Epidermal growth factor receptor (EGFR), a transmembrane glycoprotein, is overexpressed in many cancers such as head-neck, breast, prostate, and skin cancers for this reason it is a good target in cancer therapy and diagnosis. In nanobody-based cancer diagnosis and treatment, nanobodies with high affinity toward receptor (e.g. EGFR) results in effective treatment or diagnosis of cancer. In this regard, the main aim of this study is to develop a method based on molecular dynamic (MD) simulations for designing of 7D12 based nanobody with high affinity compared with wild-type nanobody. By surveying electrostatic and desolvation interactions between different residues of 7D12 and EGFR, the critical residues of 7D12 that play the main role in the binding of 7D12 to EGFR were elucidated and based on these residues, five logical variants were designed. Following the 50 ns MD simulations, pull and umbrella sampling simulation were performed for 7D12 and all its variants in complex with EGFR. Binding free energy of 7D12 (and all its variants) with EGFR was obtained by weighted histogram analysis method. According to binding free energy results, GLY101 to GLU mutation showed the highest binding affinity but this variant is unstable after 50 ns MD simulations. ALA100 to GLU mutation shows suitable binding enhancement with acceptable structural stability. Suitable position and orientation of GLU in residue 100 of 7D12 against related amino acids of EGFR formed some extra hydrogen and electrostatic interactions which resulted in binding enhancement.

  20. Mutagenesis and Teratogenesis Section

    International Nuclear Information System (INIS)

    Anon.

    1976-01-01

    Progress is reported on research with mice in the areas of radioinduced and chemical mutagenesis, cytologic studies, radiation effects on DNA synthesis, radiation effects on germ cells, mutagenicity of coal-conversion products, and others. Research on Drosophila was concerned with mutagenesis and genetics of nucleases. Studies were conducted on hamster cells with regard to cytotoxicity and mutagenicity of alkylating agents, modification of the microtubule system, protein kinase activity, and others. Research on bacteria was concerned with effects of x radiation on bacteriophage of Haemophilus influenzae, x-ray induced DNA polymerase I-directed repair synthesis in Escherichia coli, transformation by DNA polymerase II in Bacillus subtilis, and others. Research on xenopus laevis was conducted in the areas of calcium-induced cleavage of oocytes, yolk degradation in explants, and others

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

    Science.gov (United States)

    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.

  2. Classical mutagenesis in higher plants

    NARCIS (Netherlands)

    Koornneef, M.

    2002-01-01

    For a long time, mutagenesis research in plants focused on crop improvement and, especially for crop plants, opimised protocols were developed with barley being one of the favourite species. However, the interest in mutagenesis has shifted to basic plant research in the last 20 years, when the power

  3. Nevada Test Site-Directed Research, Development, and Demonstration

    International Nuclear Information System (INIS)

    Will Lewis, Compiler

    2006-01-01

    The Nevada Test Site-Directed Research, Development, and Demonstration (SDRD) program completed a very successful year of research and development activities in FY 2005. Fifty new projects were selected for funding this year, and five FY 2004 projects were brought to conclusion. The total funds expended by the SDRD program were $5.4 million, for an average per project cost of just under $100,000. Two external audits of SDRD accounting practices were conducted in FY 2005. Both audits found the program's accounting practices consistent with the requirements of DOE Order 413.2A, and one included the observation that the NTS contractor ''did an exceptional job in planning and executing year-start activities.'' Highlights for the year included: the filing of 18 invention disclosures for intellectual property generated by FY 2005 projects; programmatic adoption of 17 FY 2004 SDRD-developed technologies; participation in the tri-lab Laboratory Directed Research and Development (LDRD) and SDRD program review that was broadly attended by NTS, NNSA, LDRD, and U.S. Department of Homeland Security representatives; peer reviews of all FY 2005 projects; and the successful completion of 55 R and D projects, as presented in this report

  4. Antimicrobials, stress and mutagenesis.

    Directory of Open Access Journals (Sweden)

    Alexandro Rodríguez-Rojas

    2014-10-01

    Full Text Available Cationic antimicrobial peptides are ancient and ubiquitous immune effectors that multicellular organisms use to kill and police microbes whereas antibiotics are mostly employed by microorganisms. As antimicrobial peptides (AMPs mostly target the cell wall, a microbial 'Achilles heel', it has been proposed that bacterial resistance evolution is very unlikely and hence AMPs are ancient 'weapons' of multicellular organisms. Here we provide a new hypothesis to explain the widespread distribution of AMPs amongst multicellular organism. Studying five antimicrobial peptides from vertebrates and insects, we show, using a classic Luria-Delbrück fluctuation assay, that cationic antimicrobial peptides (AMPs do not increase bacterial mutation rates. Moreover, using rtPCR and disc diffusion assays we find that AMPs do not elicit SOS or rpoS bacterial stress pathways. This is in contrast to the main classes of antibiotics that elevate mutagenesis via eliciting the SOS and rpoS pathways. The notion of the 'Achilles heel' has been challenged by experimental selection for AMP-resistance, but our findings offer a new perspective on the evolutionary success of AMPs. Employing AMPs seems advantageous for multicellular organisms, as it does not fuel the adaptation of bacteria to their immune defenses. This has important consequences for our understanding of host-microbe interactions, the evolution of innate immune defenses, and also sheds new light on antimicrobial resistance evolution and the use of AMPs as drugs.

  5. Classical mutagenesis in higher plants

    OpenAIRE

    Koornneef, M.

    2002-01-01

    For a long time, mutagenesis research in plants focused on crop improvement and, especially for crop plants, opimised protocols were developed with barley being one of the favourite species. However, the interest in mutagenesis has shifted to basic plant research in the last 20 years, when the power of mutant approaches in combination with molecular techniques to investigate the molecular nature of the genes became fully appreciated

  6. Functional analysis of the Na+,K+/H+ antiporter PeNHX3 from the tree halophyte Populus euphratica in yeast by model-guided mutagenesis.

    Directory of Open Access Journals (Sweden)

    Liguang Wang

    Full Text Available Na+,K+/H+ antiporters are H+-coupled cotransporters that are crucial for cellular homeostasis. Populus euphratica, a well-known tree halophyte, contains six Na+/H+ antiporter genes (PeNHX1-6 that have been shown to function in salt tolerance. However, the catalytic mechanisms governing their ion transport remain largely unknown. Using the crystal structure of the Na+/H+ antiporter from the Escherichia coli (EcNhaA as a template, we built the three-dimensional structure of PeNHX3 from P. euphratica. The PeNHX3 model displays the typical TM4-TM11 assembly that is critical for ion binding and translocation. The PeNHX3 structure follows the 'positive-inside' rule and exhibits a typical physicochemical property of the transporter proteins. Four conserved residues, including Tyr149, Asn187, Asp188, and Arg356, are indentified in the TM4-TM11 assembly region of PeNHX3. Mutagenesis analysis showed that these reserved residues were essential for the function of PeNHX3: Asn187 and Asp188 (forming a ND motif controlled ion binding and translocation, and Tyr149 and Arg356 compensated helix dipoles in the TM4-TM11 assembly. PeNHX3 mediated Na+, K+ and Li+ transport in a yeast growth assay. Domain-switch analysis shows that TM11 is crucial to Li+ transport. The novel features of PeNHX3 in ion binding and translocation are discussed.

  7. Comments on mutagenesis risk estimation

    International Nuclear Information System (INIS)

    Russell, W.L.

    1976-01-01

    Several hypotheses and concepts have tended to oversimplify the problem of mutagenesis and can be misleading when used for genetic risk estimation. These include: the hypothesis that radiation-induced mutation frequency depends primarily on the DNA content per haploid genome, the extension of this concept to chemical mutagenesis, the view that, since DNA is DNA, mutational effects can be expected to be qualitatively similar in all organisms, the REC unit, and the view that mutation rates from chronic irradiation can be theoretically and accurately predicted from acute irradiation data. Therefore, direct determination of frequencies of transmitted mutations in mammals continues to be important for risk estimation, and the specific-locus method in mice is shown to be not as expensive as is commonly supposed for many of the chemical testing requirements

  8. Radiation mutagenesis of subtropic plants

    International Nuclear Information System (INIS)

    Kerkadze, I.G.

    1987-01-01

    Possibilities of expansion of subtropic plant changeability and development of new gene bank for future selection-genetic studies are detected. New trends of radiation mutagenesis of subtropic plants are formulated as results of studies during many years. A lot of mutants is subjected to sufficient tests, and concrete results are obtained with the help of these tests for definite species. Summing genetic and selection estimations of the results, it is possible to make the conclusion that mutant selection represents one of the powerful methods of preparation of productive and qualitative species of subtropic plants, which are successfully introduced into practice

  9. Nevada National Security Site: Site-Directed Research and Development (SDRD) Fiscal Year 2015 Annual Report

    Energy Technology Data Exchange (ETDEWEB)

    Bender, Howard A. [National Security Technologies, LLC. (NSTec), Mercury, NV (United States). Site-Directed Research and Development Program

    2016-04-01

    This report presents results of multiple research projects, new and ongoing, funded under the Site-Directed Research and Development Program for the Nevada National Security Site during federal fiscal year 2015. The Site's legacy capabilities in remote sensing combined with new paradigms for emergency response and consequence management help drive the need to develop advanced aerial sensor platforms. Likewise, dynamic materials science is a critical area of scientific research for which basic physics issues are still unresolved. New methods of characterizing materials in extreme states are vitally needed, and these efforts are paving the way with new knowledge. Projects selected in FY 2015 for the Exploratory Research portfolio exhibit a strong balance of NNSS mission relevance. Geoscience, seismology, and techniques for detecting underground nuclear events are still essential focus areas. Many of the project reports in the second major section of this annual report are ongoing continuations in multi-year lifecycles. Diagnostic techniques for stockpile and nuclear security science figured prominently as well, with a few key efforts coming to fruition, such as phase transition detection. In other areas, modeling efforts toward better understanding plasma focus physics has also started to pay dividends for major program needs.

  10. The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

    OpenAIRE

    Hoskins, J.T.; Zhou, Z.; Harding, P.A.

    2008-01-01

    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/Ser108/121, HB-EGF-Cys/Ser116/132, and HB-EGF-Cys/Ser134/143) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with Mr of 6.5, 21 and 24kDa were observed from lys...

  11. Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata

    OpenAIRE

    Liew, Yvonne Jing Mei; Soh, Wai Tuck; Jiemy, William Febry; Hwang, Jung Shan

    2015-01-01

    Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and teste...

  12. Beta-D-xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-Directed Mutagenesis, Alternate Substrates, and Active-site Inhibitor

    Science.gov (United States)

    Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium (SXA) is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D xylose. Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic ...

  13. Beta-D-xylosidase from Selenomonas ruminantium: role of glutamate 186 in catalysis revealed by site-directed mutagenesis, alternate substrates, and inhibitors

    Science.gov (United States)

    Beta-D-xylosidase from Selenomonas ruminantium is the best catalyst known for promoting hydrolysis of 1,4-beta-D-xylooligosaccharides, and it has potential utility in industrial saccharification processes. Kinetic parameters, kcat and kcat/Km, are more than 10-fold larger than those reported for th...

  14. Beta-D-xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-directed Mutagenesis, Alternate Substrates, and Inhibitor

    Science.gov (United States)

    Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium (SXA) is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D-xylose. Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic ...

  15. Characterization of the β-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies

    NARCIS (Netherlands)

    Alkema, Wynand B.L.; Hensgens, Charles M.H.; Kroezinga, Els H.; de Vries, Erik; Floris, René; Laan, Jan-Metske van der; Dijkstra, Bauke W.; Janssen, Dick B.

    2000-01-01

    The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme–substrate complex was determined after soaking crystals of an inactive βN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in

  16. Characterization of the beta-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies

    NARCIS (Netherlands)

    Alkema, WBL; Hensgens, CMH; Kroezinga, EH; de Vries, E; Floris, R; van der Laan, JM; Dijkstra, BW; Janssen, DB

    2000-01-01

    The binding of penicillin to penicillin acylase was studied by X-ray crystallography, The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive beta N241A penicillin acylase mutant with penicillin G, Binding of the substrate induces a conformational change,

  17. Dominating IgE-binding epitope of Bet v 1, the major allergen of birch pollen, characterized by X-ray crystallography and site-directed mutagenesis

    DEFF Research Database (Denmark)

    Spangfort, Michael D; Mirza, Osman; Ipsen, Henrik

    2003-01-01

    approach to the engineering of allergen molecules with reduced IgE binding. In this study, we describe the identification and modification of a human IgE-binding epitope based on the crystal structure of Bet v 1 in complex with the BV16 Fab' fragment. The epitope occupies approximately 10% of the molecular...... surface area of Bet v 1 and is clearly conformational. A synthetic peptide representing a sequential motif in the epitope (11 of 16 residues) did not inhibit the binding of mAb BV16 to Bet v 1, illustrating limitations in the use of peptides for B cell epitope characterization. The single amino acid...... substitution, Glu(45)-Ser, was introduced in the epitope and completely abolished the binding of mAb BV16 to the Bet v 1 mutant within a concentration range 1000-fold higher than wild type. The mutant also showed up to 50% reduction in the binding of human polyclonal IgE, demonstrating that glutamic acid 45...

  18. Dominating IgE-binding epitope of Bet v 1, the major allergen of birch pollen, characterized by X-ray crystallography and site-directed mutagenesis

    DEFF Research Database (Denmark)

    Spangfort, Michael D; Mirza, Osman; Ipsen, Henrik

    2003-01-01

    Specific allergy vaccination is an efficient treatment for allergic disease; however, the development of safer vaccines would enable a more general use of the treatment. Determination of molecular structures of allergens and allergen-Ab complexes facilitates epitope mapping and enables a rational...

  19. Crucial role of Asp408 in the proton translocation pathway of multidrug transporter AcrB: evidence from site-directed mutagenesis and carbodiimide labeling.

    Science.gov (United States)

    Seeger, Markus A; von Ballmoos, Christoph; Verrey, François; Pos, Klaas M

    2009-06-30

    The three-component AcrA/AcrB/TolC efflux system of Escherichia coli catalyzes the proton motive force-driven extrusion of a variety of cytotoxic compounds. The inner membrane pump component AcrB belongs to the resistance nodulation and cell division (RND) superfamily and is responsible for drug specificity and energy transduction of the entire tripartite efflux system. Systematic mutational analysis of titratable and polar membrane-located amino acids revealed four residues, D407, D408, K940, and, R971, to be of prime importance for AcrB function. Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, D408 was shown to specifically react with dicyclohexylcarbodiimide (DCCD) in a pH-dependent manner. The apparent pK(a) of D408 of 7.4 would enable binding and release of protons under physiological conditions. In contrast to other secondary transporters, D408 was not protected from carbodiimide modification in the presence of drugs, which supports the notion of spatially separated transport pathways for drugs and protons. This study provides evidence for a substantial role of membrane-located carboxylates as a central element of the proton translocation pathway in AcrB and other members of the RND superfamily.

  20. Engineering the cytokinin-glucoside specificity of the maize beta-D-glucosidase Zm-p60.1 using site-directed random mutagenesis

    Czech Academy of Sciences Publication Activity Database

    Filipi, T.; Mazura, P.; Janda, L.; Kiran, N.S.; Brzobohatý, Břetislav

    2012-01-01

    Roč. 74, FEB2012 (2012), s. 40-48 ISSN 0031-9422 Institutional support: RVO:68081707 Keywords : beta-Glucosidase * cis-Zeatin-O-beta-D-glucopyranoside * Cytokinin metabolism Subject RIV: BO - Biophysics Impact factor: 3.050, year: 2012

  1. An Examination by Site-Directed Mutagenesis of Putative Key Residues in the Determination of Coenzyme Specificity in Clostridial NAD+-Dependent Glutamate Dehydrogenase

    Directory of Open Access Journals (Sweden)

    Joanna Griffin

    2011-01-01

    Full Text Available Sequence and structure comparisons of various glutamate dehydrogenases (GDH and other nicotinamide nucleotide-dependent dehydrogenases have potentially implicated certain residues in coenzyme binding and discrimination. We have mutated key residues in Clostridium symbiosum NAD+-specific GDH to investigate their contribution to specificity and to enhance acceptance of NADPH. Comparisons with E. coli NADPH-dependent GDH prompted design of mutants F238S, P262S, and F238S/P262S, which were purified and assessed at pH 6.0, 7.0, and 8.0. They showed markedly increased catalytic efficiency with NADPH, especially at pH 8.0 (∼170-fold for P262S and F238S/P262S with relatively small changes for NADH. A positive charge introduced through the D263K mutation also greatly increased catalytic efficiency with NADPH (over 100-fold at pH 8 and slightly decreased activity with NADH. At position 242, “P6” of the “core fingerprint,” where NAD+- and NADP+-dependent enzymes normally have Gly or Ala, respectively, clostridial GDH already has Ala. Replacement with Gly produced negligible shift in coenzyme specificity.

  2. An Examination by Site-Directed Mutagenesis of Putative Key Residues in the Determination of Coenzyme Specificity in Clostridial NAD+-Dependent Glutamate Dehydrogenase

    OpenAIRE

    Griffin, Joanna; Engel, Paul C.

    2011-01-01

    Sequence and structure comparisons of various glutamate dehydrogenases (GDH) and other nicotinamide nucleotide-dependent dehydrogenases have potentially implicated certain residues in coenzyme binding and discrimination. We have mutated key residues in Clostridium symbiosum NAD+-specific GDH to investigate their contribution to specificity and to enhance acceptance of NADPH. Comparisons with E. coli NADPH-dependent GDH prompted design of mutants F238S, P262S, and F238S/P262S, which were purif...

  3. Dissecting the Catalytic Mechanism of Betaine - Homocysteine S-Methyltransferase by Use of Intrinsic Tryptophan Fluorescence and Site-Directed Mutagenesis

    Czech Academy of Sciences Publication Activity Database

    Castro, C.; Gratson, A. A.; Evans, J. C.; Jiráček, Jiří; Collinsová, Michaela; Ludwig, M. L.; Garrow, T. A.

    2004-01-01

    Roč. 43, č. 18 (2004), s. 5341-5351 ISSN 0006-2960 R&D Projects: GA AV ČR IAA4055302 Grant - others:NIH(US) GM16429; NIH(US) DK52501; Illinois Agricultural Experimental Station(US) ILLU-698-352 Institutional research plan: CEZ:AV0Z4055905 Keywords : BHMT * CBHcy * fluorescence Subject RIV: CE - Biochemistry Impact factor: 4.008, year: 2004

  4. Substitutions in PBP3 confer resistance to both ampicillin and extended-spectrum cephalosporins in Haemophilus parainfluenzae as revealed by site-directed mutagenesis and gene recombinants

    DEFF Research Database (Denmark)

    Wienholtz, Nanna H; Ciechanowski, Aynur Barut; Nørskov-Lauritsen, Niels

    2017-01-01

    I from clinical strains encoding four substitutions in the transpeptidase region of PBP3 conferred resistance to ampicillin, but not to cephalosporins. Introduction of ftsI from a clinical strain encoding eight substitutions conferred resistance to ampicillin, cefotaxime and ceftriaxone. MICs....../H/S in combination with V511A were resistant to ampicillin. Substitution S385T increased the MICs of third-generation cephalosporins if V511A was also present. Conclusions: Substitutions in PBP3 are sufficient to confer resistance to both ampicillin and third-generation cephalosporins in H. parainfluenzae...... . A combination of substitutions at positions Val-511 and Asn-526 confers resistance to ampicillin. Resistance to third-generation cephalosporins probably requires more than four substitutions in PBP3....

  5. Site-Directed Mutagenesis Study of an Antibiotic-Sensing Noncoding RNA Integrated into a One-Semester Project-Based Biochemistry Lab Course

    Science.gov (United States)

    Gerczei, Timea

    2017-01-01

    A laboratory sequence is described that is suitable for upper-level biochemistry or molecular biology laboratories that combines project-based and traditional laboratory experiments. In the project-based sequence, the individual laboratory experiments are thematically linked and aim to show how a bacterial antibiotic sensing noncoding RNA (the…

  6. Transglycosylation reaction catalyzed by a class V chitinase from cycad, Cycas revoluta: a study involving site-directed mutagenesis, HPLC, and real-time ESI-MS.

    Science.gov (United States)

    Taira, Toki; Fujiwara, Maho; Dennhart, Nicole; Hayashi, Hiroko; Onaga, Shoko; Ohnuma, Takayuki; Letzel, Thomas; Sakuda, Shohei; Fukamizo, Tamo

    2010-04-01

    Class V chitinase from cycad, Cycas revoluta, (CrChi-A) is the first plant chitinase that has been found to possess transglycosylation activity. To identify the structural determinants that bring about transglycosylation activity, we mutated two aromatic residues, Phe166 and Trp197, which are likely located in the acceptor binding site, and the mutated enzymes (F166A, W197A) were characterized. When the time-courses of the enzymatic reaction toward chitin oligosaccharides were monitored by HPLC, the specific activity was decreased to about 5-10% of that of the wild type and the amounts of transglycosylation products were significantly reduced by the individual mutations. From comparison between the reaction time-courses obtained by HPLC and real-time ESI-MS, we found that the transglycosylation reaction takes place under the conditions used for HPLC but not under the ESI-MS conditions. The higher substrate concentration (5 mM) used for the HPLC determination is likely to bring about chitinase-catalyzed transglycosylation. Kinetic analysis of the time-courses obtained by HPLC indicated that the sugar residue affinity of +1 subsite was strongly reduced in both mutated enzymes, as compared with that of the wild type. The IC(50) value for the inhibitor allosamidin determined by real-time ESI-MS was not significantly affected by the individual mutations, indicating that the state of the allosamidin binding site (from -3 to -1 subsites) was not changed in the mutated enzymes. We concluded that the aromatic side chains of Phe166 and Trp197 in CrChi-A participate in the transglycosylation acceptor binding, thus controlling the transglycosylation activity of the enzyme. Copyright 2009 Elsevier B.V. All rights reserved.

  7. Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile: cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues.

    OpenAIRE

    Kobayashi, M; Izui, H; Nagasawa, T; Yamada, H

    1993-01-01

    Indole-3-acetic acid is the major auxin in most plants. In Cruciferae, including Brassicaceae, indole-3-acetic acid is synthesized from indole-3-acetonitrile by nitrilase, after indole-3-acetonitrile is formed from tryptophan via indole-3-acetaldoxime or indole glycosinolates as the intermediate. We cloned and sequenced the gene for nitrilase (EC 3.5.5.1), which catalyzes the hydrolysis of indole-3-acetonitrile to indole-3-acetic acid, from Alcaligenes faecalis JM3. The amino acid sequence de...

  8. Probing the role of helix α1 in the acid-tolerance and thermal stability of the Streptomyces sp. SK Glucose Isomerase by site-directed mutagenesis.

    Science.gov (United States)

    Hajer, Ben Hlima; Dorra, Zouari Ayadi; Monia, Mezghani; Samir, Bejar; Nushin, Aghajari

    2014-03-10

    In order to investigate the role of helix α1 in the different biochemical properties between class I and class II Glucose Isomerases, a histidine and a phenylalanine residue were inserted at position 17 and 19 of Streptomyces sp. SK Glucose Isomerase (SKGI). In addition, W16 was substituted by a histidine. The H17/F19 insertion displaced the optimal pH of SKGI from 6.5 to 7-8 and slightly decreased the thermostability. As for the W16H mutant, a shift in optimal pH of SKGI from 6.5 to 6 was observed along with a decrease in the enzyme thermostability at 85°C with a half-life time reduced twice compared to the wild-type enzyme. Three-dimensional structure analysis suggested that the insertion of a histidine at position 17 results in the formation of new hydrogen bond with D287, thereby preventing it from deprotonating the O2 hydroxyl of the sugar at low pH, while the substitution W16H induced opposite effect by preventing hydrogen bond formation between D287 and W16 and thereby probably facilitating the hydrogen transfer during the isomerization reaction. The findings highlight the essential role of helix α1, which bears the three introduced mutations, in the acid-tolerance and the thermostability of SKGI and of glucose isomerases in general. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Increased L-arginine Production by Site-directed Mutagenesis of N-acetyl-L-glutamate Kinase and proB Gene Deletion in Corynebacterium crenatum.

    Science.gov (United States)

    Zhang, Bin; Wan, Fang; Qiu, Yu Lou; Chen, Xue Lan; Tang, Li; Chen, Jin Cong; Xiong, Yong Hua

    2015-12-01

    In Corynebacterium crenatum, the adjacent D311 and D312 of N-acetyl-L-glutamate kinase (NAGK), as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine, were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK. NAGK enzyme expression was evaluated using a plasmid-based method. Homologous recombination was employed to eliminate the proB. The IC50 and enzyme activity of NAGK M4, in which the D311R and D312R amino acid substitutions were combined with the previously reported E19R and H26E substitutions, were 3.7-fold and 14.6% higher, respectively, than those of the wild-type NAGK. NAGK M4 was successfully introduced into the C. crenatum MT genome without any genetic markers; the L-arginine yield of C. crenatum MT-M4 was 26.2% higher than that of C. crenatum MT. To further improve upon the L-arginine yield, we constructed the mutant C. crenatum MT-M4 proB. The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield. After L-proline was added to the medium at 10 mmol/L, the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation, approximately 70.1% higher than the yield attained using C. crenatum MT. Feedback inhibition of L-arginine on NAGK in C. crenatum is clearly alleviated by the M4 mutation of NAGK, and deletion of the proB in C. crenatum from MT to M4 results in a significant increase in arginine production. Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  10. Site-directed mutagenesis of Arg58 and Asp86 of elongation factor Tu from Escherichia coli: effects on the GTPase reaction and aminoacyl-tRNA binding

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Clark, Brian F. C.

    1996-01-01

    Elongation factor Tu from Escherichia coli was mutated separately at positions Asp86 and Arg58, in order to shed light both on the GTPase mechanism of elongation factor Tu and on the binding of aminoacyl-tRNA. In addition, the binding of guanine nucleotides was investigated by determination...

  11. Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds: alteration of pyridine nucleotide binding by a single amino acid substitution.

    Science.gov (United States)

    Kobori, T; Sasaki, H; Lee, W C; Zenno, S; Saigo, K; Murphy, M E; Tanokura, M

    2001-01-26

    The crystal structure of a major oxygen-insensitive nitroreductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an alpha + beta-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent flavin reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with the V. harveyi flavin reductase and the NADPH-dependent nitro/flavin reductase of Bacillus subtilis, residues Arg(203) and Arg(208) of the loop region between helices I and J in the vicinity of the catalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in the K(m) value for NADPH indicating that the side chain of Arg(203) plays a key role in binding NADPH possibly to interact with the 2'-phosphate group.

  12. Molecular Insights into the Coding Region Determinant-binding Protein-RNA Interaction through Site-directed Mutagenesis in the Heterogeneous Nuclear Ribonucleoprotein-K-homology Domains*

    Science.gov (United States)

    Barnes, Mark; van Rensburg, Gerrit; Li, Wai-Ming; Mehmood, Kashif; Mackedenski, Sebastian; Chan, Ching-Man; King, Dustin T.; Miller, Andrew L.; Lee, Chow H.

    2015-01-01

    The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function. PMID:25389298

  13. Identification of the catalytic residues of alpha-amino acid ester hydrolase from Acetobacter turbidans by labeling and site-directed mutagenesis

    NARCIS (Netherlands)

    Polderman - Tijmes, Jolanda j.; Jekel, Peter A.; Jeronimus-Stratingh, CM; Bruins, Andries P.; van der Laan, Jan-Metske; Sonke, Theo; Janssen, Dick B.

    2002-01-01

    The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in beta-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also

  14. Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis

    DEFF Research Database (Denmark)

    Madec, Edwige; Stensballe, Allan; Kjellström, Sven

    2003-01-01

    We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing...

  15. Site-directed mutagenesis of surfactant protein A reveals dissociation of lipid aggregation and lipid uptake by alveolar type II cells.

    Science.gov (United States)

    Tsunezawa, W; Sano, H; Sohma, H; McCormack, F X; Voelker, D R; Kuroki, Y

    1998-09-08

    Surfactant protein A (SP-A) binds to dipalmitoylphosphatidylcholine (DPPC) and induces phospholipid vesicle aggregation. It also regulates the uptake and secretion of surfactant lipids by alveolar type II cells. We introduced the single mutations Glu195-->Gln (rE195Q), Lys201-->Ala (rK201A) and Lys203-->Ala (rK203A) for rat SP-A, Arg199-->Ala (hR199A) and Lys201-->Ala (hK201A) for human SP-A, and the triple mutations Arg197, Lys201 and Lys203-->Ala (rR197A/K201A/K203A) for rat SP-A, into cDNAs for SP-A, and expressed the recombinant proteins using baculovirus vectors. All recombinant proteins avidly bound to DPPC liposomes. rE195Q, rK201A, rK203A, hR199A and hK201A function with activity comparable to wild type SP-A. Although rR197A/K201A/K203A was a potent inducer of phospholipid vesicle aggregation, it failed to stimulate lipid uptake. rR197A/K201A/K203A was a weak inhibitor for lipid secretion and did not competed with rat [125I]SP-A for receptor occupancy. From these results, we conclude that Lys201 and Lys203 of rat SP-A, and Arg199 and Lys201 of human SP-A are not individually critical for the interaction with lipids and type II cells, and that Glu195 of rat SP-A can be replaced with Gln without loss of SP-A functions. This study also demonstrates that the SP-A-mediated lipid uptake is not directly correlated with phospholipid vesicle aggregation, and that specific interactions of SP-A with type II cells are involved in the lipid uptake process.

  16. Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis

    DEFF Research Database (Denmark)

    Madec, Edwige; Stensballe, Allan; Kjellström, Sven

    2003-01-01

    the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high...

  17. Direct random insertion mutagenesis of Helicobacter pylori

    NARCIS (Netherlands)

    de Jonge, Ramon; Bakker, Dennis; van Vliet, Arnoud H. M.; Kuipers, Ernst J.; Vandenbroucke-Grauls, Christina M. J. E.; Kusters, Johannes G.

    2003-01-01

    Random insertion mutagenesis is a widely used technique for the identification of bacterial virulence genes. Most strategies for random mutagenesis involve cloning in Escherichia coli for passage of plasmids or for phenotypic selection. This can result in biased selection due to restriction or

  18. Laboratory of Mutagenesis and DNA Repair

    International Nuclear Information System (INIS)

    2000-01-01

    Full text: Two main lines of research were continued: the first one concerned the mechanisms controlling the fidelity of DNA replication in Escherichia coli; the second concerned cellular responses of Saccharomyces cerevisiae to DNA damaging agents. We have been investigating the question whether during chromosomal DNA replication in Escherichia coli the two DNA strands may be replicated with differential accuracy. To address this question we set up a new system that allows the examination of mutagenesis either of the leading strand or the lagging strand. Our results suggest that the lagging strand replication of the E. coli chromosome may be more accurate than leading strand replication. More recently, we studied mutagenesis of the two strands in recA730 strains which exhibit constitutive expression of the SOS system. Our results clearly indicate that in recA730 strains there is a significant difference in the fidelity of replication between the two replicating strands. Based on our data we propose a model describing a possible mechanism of SOS mutagenesis. To get more insight into cellular responses to DNA damage we have isolated several novel genes of S. cerevisiae, the transcription of which is induced by DNA lesions. Main effort was concentrated on the characterization of the DIN7 gene. We found that Din7p specifically affects the metabolism of mitochondrial DNA (mtDNA). The elevated level of Din7p results in an increased frequency of mitochondrial petite mutants, as well as in a higher frequency of mitochondrial point mutations. Din7p affects also the stability of microsatellite sequences present in the mitochondrial genome. As expected, Din7p was found to be located in mitochondria. In another project, we found that the DIN8 gene isolated in our laboratory is identical with the UMP1 gene encoding a chaperone-like protein involved in 20S proteasome maturation. Interestingly, induction of UMP1 expression in response to DNA damage is subject to regulation

  19. Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

    Energy Technology Data Exchange (ETDEWEB)

    Couto, Sheila G. [Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense 400, C.P. 369, 13560-970, Sao Carlos, SP (Brazil); Grupo de Biofisica e Fisica Aplicada a Medicina, Instituto de Fisica, Universidade Federal de Goias, Campus Samambaia, C.P. 131, 74001-970, Goiania, GO (Brazil); Cristina Nonato, M. [Laboratorio de Cristalografia de Proteinas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo, Av. do Cafe S/N, 14040-903, Ribeirao Preto, SP (Brazil); Costa-Filho, Antonio J., E-mail: ajcosta@ffclrp.usp.br [Instituto de Fisica de Sao Carlos, Universidade de Sao Paulo, Av. Trabalhador Sao-carlense 400, C.P. 369, 13560-970, Sao Carlos, SP (Brazil); Departamento de Fisica, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Av. Bandeirantes 3900, 14040-901, Ribeirao Preto, SP (Brazil)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer EcDHODH is a membrane-associated enzyme and a promising target for drug design. Black-Right-Pointing-Pointer Enzyme's N-terminal extension is responsible for membrane association. Black-Right-Pointing-Pointer N-terminal works as a molecular lid regulating access to the protein interior. -- Abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

  20. Cellular components required for mutagenesis

    International Nuclear Information System (INIS)

    Elledge, S.J.; Perry, K.L.; Krueger, J.H.; Mitchell, B.B.; Walker, G.C.

    1983-01-01

    We have cloned the umuD and umuC genes of Escherichia coli and have shown that they code for two proteins of 16,000 and 45,000 daltons respectively; the two genes are organized in an operon that is repressed by the LexA protein. Similarly, we have shown that the mucA and mucB genes of the mutagenesis-enhancing plasmid pKM101 code for proteins of 16,000 and 45,000 daltons respectively and, like umuD/C, the genes are organized in an operon. Preliminary sequencing studies have indicated that the umuD/C and mucA/B loci are approximately 50% homologous at both the nucleic acid and deduced protein sequence levels and that the umuD gene is preceeded by two putative LexA binding sites separated by 4 basepairs. Like umuD/C, the mucA/B genes of pKM101 are induced by DNA damage and are repressed by LexA. In addition to inducing recA + lexA + -regulated din genes, DNA damaging agents such as uv and nalidixic acid also induce the heat shock proteins GroEL and DnaK in an htpR-dependent fashion. 22 references, 1 figure, 1 table

  1. Characterization of the Pathogenicity of Streptococcus intermedius TYG1620 Isolated from a Human Brain Abscess Based on the Complete Genome Sequence with Transcriptome Analysis and Transposon Mutagenesis in a Murine Subcutaneous Abscess Model.

    Science.gov (United States)

    Hasegawa, Noriko; Sekizuka, Tsuyoshi; Sugi, Yutaka; Kawakami, Nobuhiro; Ogasawara, Yumiko; Kato, Kengo; Yamashita, Akifumi; Takeuchi, Fumihiko; Kuroda, Makoto

    2017-02-01

    Streptococcus intermedius is known to cause periodontitis and pyogenic infections in the brain and liver. Here we report the complete genome sequence of strain TYG1620 (genome size, 2,006,877 bp; GC content, 37.6%; 2,020 predicted open reading frames [ORFs]) isolated from a brain abscess in an infant. Comparative analysis of S. intermedius genome sequences suggested that TYG1620 carries a notable type VII secretion system (T7SS), two long repeat regions, and 19 ORFs for cell wall-anchored proteins (CWAPs). To elucidate the genes responsible for the pathogenicity of TYG1620, transcriptome analysis was performed in a murine subcutaneous abscess model. The results suggest that the levels of expression of small hypothetical proteins similar to phenol-soluble modulin β1 (PSMβ1), a staphylococcal virulence factor, significantly increased in the abscess model. In addition, an experiment in a murine subcutaneous abscess model with random transposon (Tn) mutant attenuation suggested that Tn mutants with mutations in 212 ORFs in the Tn mutant library were attenuated in the murine abscess model (629 ORFs were disrupted in total); the 212 ORFs are putatively essential for abscess formation. Transcriptome analysis identified 37 ORFs, including paralogs of the T7SS and a putative glucan-binding CWAP in long repeat regions, to be upregulated and attenuated in vivo This study provides a comprehensive characterization of S. intermedius pathogenicity based on the complete genome sequence and a murine subcutaneous abscess model with transcriptome and Tn mutagenesis, leading to the identification of pivotal targets for vaccines or antimicrobial agents for the control of S. intermedius infections. Copyright © 2017 American Society for Microbiology.

  2. DNA repair and mutagenesis of singlestranded bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

    Doubleday, O.P.; Brandenburger, A.; Wagner, R. Jr.; Radman, M. (Brussels Univ. (Belgium)); Godson, G.N.

    1981-01-01

    Virtually all radiation-induced mutagenesis is believed to result from an error-prone repair activity (SOS repair) and to involve mutations occurring both at the site of radiation-induced lesions (targeted mutations) and in undamaged DNA (untargeted mutations). To examine the relative contributions of targeted and untargeted mutations to ..gamma.. and ultraviolet (UV) radiation mutagenesis we have determined the DNA sequences of 174 M13 revertant phages isolated from stocks of irradiated or unirradiated amber mutants grown in irradiated or unirradiated host bacteria. We have detected no obvious specificity of mutagenesis and find no evidence of a predominance of targeted mutations associated with either UV- or ..gamma..-irradiation of the phages or with the induction of the host SOS repair system. In particular, pyrimidine dimers do not appear to be the principal sites of UV-induced bare substitution mutagenesis, suggesting that such UV-induced mutagenesis may be untargeted or occur at sites of lesions other than pyrimidine dimers.

  3. Estimations of On-site Directional Wave Spectra from Measured Ship Responses

    DEFF Research Database (Denmark)

    Nielsen, Ulrik Dam

    2006-01-01

    include an quivalence of energy in the governing equations and, as regards the parametric concept, a frequency dependent spreading of the waves is introduced. The paper includes an extensive analysis of full-scale measurements for which the directional wave spectra are estimated by the two ship response......In general, two main concepts can be applied to estimate the on-site directional wave spectrum on the basis of ship response measurements: 1) a parametric method which assumes the wave spectrum to be composed by parameterised wave spectra, or 2) a non-parametric method where the directional wave...

  4. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

    Directory of Open Access Journals (Sweden)

    Weir Jerry P

    2007-05-01

    Full Text Available Abstract Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2 BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.

  5. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes.

    Science.gov (United States)

    Schmeisser, Falko; Weir, Jerry P

    2007-05-14

    Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.

  6. Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms

    Energy Technology Data Exchange (ETDEWEB)

    Ford, Nicole R.; Hecht, Karen A.; Hu, Dehong; Orr, Galya; Xiong, Yijia; Squier, Thomas; Rorrer, Gregory L.; Roesijadi, Guritno

    2016-01-08

    The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins incorporating a tetracysteine tag for site-directed labeling with biarsenical affinity probes and either EGFP or single chain antibody to test colocalization of probes with the EGFP-tagged recombinant protein or binding of biosilica-immobilized antibodies to large and small molecule antigens, respectively. Site-directed labeling with the biarsenical probes demonstrated colocalization with EGFP-encoded proteins in nascent and mature biosilica, supporting their use in studying biosilica maturation. Isolated biosilica transformed with a single chain antibody against either the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT) effectively bound the respective antigens. A marked increase in fluorescence lifetime of the TNT surrogate Alexa Fluor 555-trinitrobenzene reflected the high binding specificity of the transformed isolated biosilica. These results demonstrated the potential use of biosilica-immobilized single chain antibodies as binders for large and small molecule antigens in sensing and therapeutics.

  7. Crystallization and preliminary crystallographic studies of human septin 1 with site-directed mutations

    International Nuclear Information System (INIS)

    Hu, Hao; Yu, Wen-bo; Li, Shu-xing; Ding, Xiang-ming; Yu, Long; Bi, Ru-Chang

    2006-01-01

    The homogeneity of septin 1 has been improved by site-directed mutation of serine residues and only a small alteration in the secondary structure is observed to arise from the mutations. Crystals of the septin 1 mutant were grown and diffraction data were collected to 2.5 Å resolution. Septin 1 is a member of an evolutionarily conserved family of GTP-binding and filament-forming proteins named septins, which function in diverse processes including cytokinasis, vesicle trafficking, apoptosis, remodelling of the cytoskeleton, infection, neurodegeneration and neoplasia. Human septin 1 has been expressed and purified, but suffers from severe aggregation. Studies have shown that septin 1 with site-directed mutations of five serine residues (Ser19, Ser206, Ser307, Ser312 and Ser315) has a much lower degree of aggregation and better structural homogeneity and that the mutations cause only slight perturbations in the secondary structure of septin 1. This septin 1 mutant was crystallized and diffraction data were collected to 2.5 Å resolution. The space group is P422, with unit-cell parameters a = b = 106.028, c = 137.852 Å

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

    Science.gov (United States)

    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.

  9. Photodynamic action of the methylene blue: mutagenesis and sinergism

    International Nuclear Information System (INIS)

    Capella, M.A.M.

    1988-01-01

    Two aspects of photodynamic therapy were studied: the associated mutagenesis and the interactions with physical agents, in order to increase its biological effects. The photodynamic action with methylene blue in the mutagenesis and sinergism is studied. (L.M.J.)

  10. Stationary-State Mutagenesis in Escherichia coli

    Indian Academy of Sciences (India)

    Stationary-phase mutagenesis in nondividing E. coli cells exposed to a nonlethal stress was, a few years ago, claimed to be a likely case of a Lamarckian mechanism capable of producing exclusively useful mutations in a directed manner. After a heated debate over the last decade it now appears to involve a Darwinian ...

  11. Studies on radioisotope mutagenesis in mammals

    International Nuclear Information System (INIS)

    Reddi, O.S.; Naidu, N.V.; Reddy, P.P.

    1974-01-01

    Studies on radioisotope mutagenesis are important from the point of view of the possible genetic hazards of their increasing use in medical and industrial applications and their concentration in man through environmental contamination. This paper reviews a series of studies undertaken on the genetic consequences of internal administation of certain selected radioisotopes, namely, 32 P, 131 I and 90 Sr in mammalian systems. (author)

  12. Complex epidemiological approach to human mutagenesis

    International Nuclear Information System (INIS)

    Czeizel, A.

    1980-01-01

    The main characteristics of the epidemiological approach are summarised and the criteria discussed for the adoption of this approach for the detection of human mutagenesis. Mutation monitoring systems are described and results of epidemiological studies of higher risk populations are presented. (C.F.)

  13. Seed mutagenesis in Portulaca grandiflora (Hook)

    International Nuclear Information System (INIS)

    Bennani, F.; Rossi-Hassani, B.D.

    2001-01-01

    Betalain pigments have been used as natural additives. Despite their importance, the biochemistry and genetics of betalain synthesis remain relatively undetermined. Portulaca grandiflora represents an ideal material for genetic analysis. In the present work, seed mutagenesis was examined with a view to enhance the chance of detection of new genetic markers in this species

  14. Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Sandra Witz

    Full Text Available Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.

  15. Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis.

    Science.gov (United States)

    Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M Joanne; Möhlmann, Torsten

    2014-01-01

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.

  16. Structure-Function Relationship of a Plant NCS1 Member – Homology Modeling and Mutagenesis Identified Residues Critical for Substrate Specificity of PLUTO, a Nucleobase Transporter from Arabidopsis

    Science.gov (United States)

    Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M. Joanne; Möhlmann, Torsten

    2014-01-01

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. PMID:24621654

  17. Structure-function relationship of a plant NCS1 member - Homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from arabidopsis

    KAUST Repository

    Witz, Sandra

    2014-03-12

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. 2014 Witz et al.

  18. Estimations of On-site Directional Wave Spectra from Measured Ship Responses

    DEFF Research Database (Denmark)

    Nielsen, Ulrik Dam

    2006-01-01

    In general, two main concepts can be applied to estimate the on-site directional wave spectrum on the basis of ship response measurements: 1) a parametric method which assumes the wave spectrum to be composed by parameterised wave spectra, or 2) a non-parametric method where the directional wave...... spectrum is found directly as the values in a completely discretised frequency-directional domain without a priori assumptions on the spectrum. The paper outlines the theory of these two concepts, and it is shown how to deal with the speed-of-advance problem for operating ships. In addition, the methods...... include an quivalence of energy in the governing equations and, as regards the parametric concept, a frequency dependent spreading of the waves is introduced. The paper includes an extensive analysis of full-scale measurements for which the directional wave spectra are estimated by the two ship response...

  19. Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example

    Directory of Open Access Journals (Sweden)

    Michael Katzberg

    2010-04-01

    Full Text Available The synthesis of pharmaceuticals and catalysts more and more relies on enantiopure chiral building blocks. These can be produced in an environmentally benign and efficient way via bioreduction of prochiral ketones catalyzed by dehydrogenases. A productive source of these biocatalysts is the yeast Saccharomyces cerevisiae, whose genome also encodes a reductase catalyzing the sequential reduction of the γ-diketone 2,5-hexanedione furnishing the diol (2S,5S-hexanediol and the γ-hydroxyketone (5S-hydroxy-2-hexanone in high enantio- as well as diastereoselectivity (ee and de >99.5%. This enzyme prefers NADPH as the hydrogen donating cofactor. As NADH is more stable and cheaper than NADPH it would be more effective if NADH could be used in cell-free bioreduction systems. To achieve this, the cofactor binding site of the dehydrogenase was altered by site-directed mutagenesis. The results show that the rational approach based on a homology model of the enzyme allowed us to generate a mutant enzyme having a relaxed cofactor preference and thus is able to use both NADPH and NADH. Results obtained from other mutants are discussed and point towards the limits of rationally designed mutants.

  20. Mutagenesis in bacteriophage T 7. 2

    International Nuclear Information System (INIS)

    Meyer, M.; Witte, W.

    1976-01-01

    UV induced mutagenesis of bacteriophage T 7 was investigated by using a forward mutation system (host range system) and a back mutation system (amber system). The results indicate a dependence of mutation of T 7 after UV irradiation only on the rec gene controlled functions of the bacterial host. The functions controlled by pol and uvr genes have no influence. Among other types of mutations UV irradiation leads to transitions from AT to GC. (author)

  1. Expression, characterization, and site-directed mutation of a multiple herbicide-resistant acetohydroxyacid synthase (rAHAS) from Pseudomonas sp. Lm10.

    Science.gov (United States)

    Lang, Zhi-Fei; Shen, Jing-Jing; Cai, Shu; Zhang, Jun; He, Jian; Li, Shun-Peng

    2011-08-01

    A multiple herbicide-resistant acetohydroxyacid synthase (rAHAS) gene was cloned from Pseudomonas sp. Lm10. Sequence analysis showed that the rAHAS regulatory subunit was identical to that of Pseudomonas putida KT2440 (sensitive AHAS, sAHAS), whereas six different sites [H134→N (rAHAS→sAHAS), A135→P, S136→T, I210→V, F264→Y, and S486→W] were found in the catalytic subunit. The rAHAS and sAHAS were over expressed, purified and characterized. rAHAS showed higher resistance to four kinds of AHAS-inhibitor herbicides than sAHAS. The resistance factor of rAHAS was 56.0-fold, 12.6-fold, 6.5-fold, and 9.2-fold as compared with sAHAS when metsulfuron-methyl, imazethapyr, flumetsulam, and pyriminobac-methyl used as inhibitor, respectively. The specific activity of rAHAS was lower than that of sAHAS and the K (m) value of rAHAS for pyruvate was approximately onefold higher than the corresponding value for sAHAS. Data from site-directed mutagenesis demonstrated that alteration at A135, F264, and S486 resulted in resistance reduction, while the mutation at H134, S136, and I210 has little effect on the resistance. A135 was mainly responsible for resistance to imidazolinone; F264 conferred resistance to sulfonylurea and triazolopyrimidine sulfonamide; and S486 showed multiple herbicides resistance to the four herbicides.

  2. Nevada Test Site-Directed Research and Development FY 2010 Annual Report

    Energy Technology Data Exchange (ETDEWEB)

    Howard Bender, comp.

    2011-04-04

    This annual report of the Site-Directed Research and Development (SDRD) program represents the highly significant R&D accomplishments conducted during fiscal year 2010. This year was noteworthy historically, as the Nevada Test Site was renamed to the Nevada National Security Site (NNSS). This change not only recognizes how the site's mission has evolved, but also heralds a future of new challenges and opportunities for the NNSS. In many ways, since its inception in 2002, the SDRD program has helped shape that evolving mission. As we approach 2012, SDRD will also mark a milestone, having completed its first full decade of innovative R&D in support of the site and national security. The program continues to fund advanced science and technology development across traditional Department of Energy (DOE) nuclear security areas such as stockpile stewardship and non-proliferation while also supporting Department of Homeland Security (DHS) needs, and specialized work for government agencies like the Department of Defense (DoD) and others. The NNSS will also contribute technologies in the areas of treaty verification and monitoring, two areas of increasing importance to national security. Keyed to the NNSS's broadened scope, the SDRD program will continue to anticipate and advance R&D projects that will help the NNSS meet forthcoming challenges.

  3. Pathogen corruption and site-directed recombination at a plant disease resistance gene cluster.

    Science.gov (United States)

    Nagy, Ervin D; Bennetzen, Jeffrey L

    2008-12-01

    The Pc locus of sorghum (Sorghum bicolor) determines dominant sensitivity to a host-selective toxin produced by the fungal pathogen Periconia circinata. The Pc region was cloned by a map-based approach and found to contain three tandemly repeated genes with the structures of nucleotide binding site-leucine-rich repeat (NBS-LRR) disease resistance genes. Thirteen independent Pc-to-pc mutations were analyzed, and each was found to remove all or part of the central gene of the threesome. Hence, this central gene is Pc. Most Pc-to-pc mutations were associated with unequal recombination. Eight recombination events were localized to different sites in a 560-bp region within the approximately 3.7-kb NBS-LRR genes. Because any unequal recombination located within the flanking NBS-LRR genes would have removed Pc, the clustering of cross-over events within a 560-bp segment indicates that a site-directed recombination process exists that specifically targets unequal events to generate LRR diversity in NBS-LRR loci.

  4. Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation.

    Directory of Open Access Journals (Sweden)

    Aerin Yoon

    Full Text Available For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv, we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7 and two VH (H13 and H16 mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on cysteine is active and their binding activity is retained after the conjugation process.

  5. Nevada Test Site-Directed Research, Development, and Demonstration. FY2005 report

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Will [comp.

    2006-09-01

    The Nevada Test Site-Directed Research, Development, and Demonstration (SDRD) program completed a very successful year of research and development activities in FY 2005. Fifty new projects were selected for funding this year, and five FY 2004 projects were brought to conclusion. The total funds expended by the SDRD program were $5.4 million, for an average per project cost of just under $100,000. Two external audits of SDRD accounting practices were conducted in FY 2005. Both audits found the program's accounting practices consistent with the requirements of DOE Order 413.2A, and one included the observation that the NTS contractor ''did an exceptional job in planning and executing year-start activities.'' Highlights for the year included: the filing of 18 invention disclosures for intellectual property generated by FY 2005 projects; programmatic adoption of 17 FY 2004 SDRD-developed technologies; participation in the tri-lab Laboratory Directed Research and Development (LDRD) and SDRD program review that was broadly attended by NTS, NNSA, LDRD, and U.S. Department of Homeland Security representatives; peer reviews of all FY 2005 projects; and the successful completion of 55 R&D projects, as presented in this report.

  6. Nevada Test Site-Directed Research and Development: FY 2006 Report

    International Nuclear Information System (INIS)

    Wil Lewis, editor

    2007-01-01

    The Nevada Test Site Directed Research and Development (SDRD) program completed its fifth successful year of research and development activities in FY 2006. Forty new projects were selected for funding this year, and ten FY 2005 projects were brought to conclusion. The total funds expended by the SDRD program were $6 million, for an average per-project cost of $120 thousand. Beginning in May, 2006 programmatic burden rates were applied to SDRD project costs. An external audit conducted in September 2006 verified that appropriate accounting practices were applied to the SDRD program. Highlights for the year included: the filing of 27 invention disclosures for intellectual property generated by FY 2006 projects; programmatic adoption of four FY 2005 SDRD-developed technologies; participation in the tri-Lab Laboratory Directed Research and Development (LDRD) and SDRD program review that was broadly attended by NTS, NNSA, LDRD, and U.S. Department of Homeland Security representatives; peer reviews of all FY 2006 projects; and the successful completion of 50 R and D projects, as presented in this report

  7. Nevada Natonal Security Site-Directed Research and Development FY 2010 Annual Report

    International Nuclear Information System (INIS)

    Bender, Howard

    2011-01-01

    This annual report of the Site-Directed Research and Development (SDRD) program represents the highly significant R and D accomplishments conducted during fiscal year 2010. This year was noteworthy historically, as the Nevada Test Site was renamed to the Nevada National Security Site (NNSS). This change not only recognizes how the site's mission has evolved, but also heralds a future of new challenges and opportunities for the NNSS. In many ways, since its inception in 2002, the SDRD program has helped shape that evolving mission. As we approach 2012, SDRD will also mark a milestone, having completed its first full decade of innovative R and D in support of the site and national security. The program continues to fund advanced science and technology development across traditional Department of Energy (DOE) nuclear security areas such as stockpile stewardship and non-proliferation while also supporting Department of Homeland Security (DHS) needs, and specialized work for government agencies like the Department of Defense (DoD) and others. The NNSS will also contribute technologies in the areas of treaty verification and monitoring, two areas of increasing importance to national security. Keyed to the NNSS's broadened scope, the SDRD program will continue to anticipate and advance R and D projects that will help the NNSS meet forthcoming challenges.

  8. Nevada National Security Site. Site-Directed Research and Development FY 2011 Annual Report

    International Nuclear Information System (INIS)

    Bender, Howard

    2012-01-01

    This fiscal year 2011 annual report of the Site-Directed Research and Development program, the 10th anniversary edition, recognizes a full decade of innovative R and D accomplishments in support of the Nevada National Security Site (NNSS). Last year the NNSS itself was renamed to reflect a diversifying mission, and our R and D program has contributed significantly to shape emerging missions that will continue to evolve. New initiatives in stockpile stewardship science, nonproliferation, and treaty verification and monitoring have had substantial successes in FY 2011, and many more accomplishments are expected. SDRD is the cornerstone on which many of these initiatives rest. Historically supporting our main focus areas, SDRD is also building a solid foundation for new, and non-traditional, emerging national security missions. The program continues its charter to advance science and technology for a broad base of agencies including the U.S. Department of Energy (DOE), U.S. Department of Defense (DoD), U.S. Department of Homeland Security (DHS), and many others.

  9. Nevada National Security Site-Directed Research and Development FY 2011 Annual Report

    Energy Technology Data Exchange (ETDEWEB)

    Howard Bender, comp.

    2012-04-25

    This fiscal year 2011 annual report of the Site-Directed Research and Development program, the 10th anniversary edition, recognizes a full decade of innovative R&D accomplishments in support of the Nevada National Security Site (NNSS). Last year the NNSS itself was renamed to reflect a diversifying mission, and our R&D program has contributed significantly to shape emerging missions that will continue to evolve. New initiatives in stockpile stewardship science, nonproliferation, and treaty verification and monitoring have had substantial successes in FY 2011, and many more accomplishments are expected. SDRD is the cornerstone on which many of these initiatives rest. Historically supporting our main focus areas, SDRD is also building a solid foundation for new, and non-traditional, emerging national security missions. The program continues its charter to advance science and technology for a broad base of agencies including the U.S. Department of Energy (DOE), U.S. Department of Defense (DoD), U.S. Department of Homeland Security (DHS), and many others.

  10. Role of radiation mutagenesis in cotton selection

    International Nuclear Information System (INIS)

    Mamedov, K.M.; Shamaeva, N.N.

    1987-01-01

    Experimantal mutagenesis in combination with the classical methods: hybridization and selection, are shown to be one of the effective methods for developing new species of cotton plants. Taking into account the character of mutant difference inheritance during hybridization of lines as well as the degree of correlative bonds, 10 most perspective lines from 198 ones are separated. They differ from the initial species by a complex of favorable hereditary changes according to the quantitative selectively useful features, that makes them advantageous for application of the existing ones and for the development of new species of fine-fiber cotton plants

  11. Nevada Test Site-Directed Research and Development, FY 2007 Report

    Energy Technology Data Exchange (ETDEWEB)

    Wil Lewis, editor

    2008-02-20

    The Nevada Test Site-Directed Research and Development (SDRD) program completed a very successful year of research and development activities in FY 2007. Twenty-nine new projects were selected for funding this year, and eight projects started in FY 2006 were brought to conclusion. The total funds expended by the SDRD program were $5.67 million, for an average per-project cost of $153 thousand. An external audit conducted in September 2007 verified that appropriate accounting practices were applied to the SDRD program. Highlights for the year included: programmatic adoption of 8 SDRD-developed technologies; the filing of 9 invention disclosures for innovation evolving from SDRD projects; participation in the tri-Lab Laboratory Directed Research and Development (LDRD) and SDRD Symposium that was broadly attended by Nevada Test Site (NTS), National Nuclear Security Administration (NNSA), LDRD, U.S. Department of Homeland Security (DHS), and U.S. Department of Defense (DoD) representatives; peer reviews of all FY 2007 projects; and the successful completion of 37 R&D projects, as presented in this report. In response to a company-wide call, authors throughout the NTS complex submitted 182 proposals for FY 2007 SDRD projects. The SDRD program has seen a dramatic increase in the yearly total of submitted proposals--from 69 in FY 2002 to 182 this year--while the number of projects funded has actually decreased from a program high of 57 in FY 2004. The overall effect of this trend has helped ensure an increasingly competitive program that benefited from a broader set of innovative ideas, making project selection both challenging and rewarding. Proposals were evaluated for technical merit, including such factors as innovation, probability of success, potential benefit, and mission applicability. Authors and reviewers benefited from the use of a shortfalls list entitled the 'NTS Technology Needs Assessment' that was compiled from NTS, National Weapons Laboratory

  12. Conformational change in full-length mouse prion: A site-directed spin-labeling study

    International Nuclear Information System (INIS)

    Inanami, Osamu; Hashida, Shukichi; Iizuka, Daisuke; Horiuchi, Motohiro; Hiraoka, Wakako; Shimoyama, Yuhei; Nakamura, Hideo; Inagaki, Fuyuhiko; Kuwabara, Mikinori

    2005-01-01

    The structure of the mouse prion (moPrP) was studied using site-directed spin-labeling electron spin resonance (SDSL-ESR). Since a previous NMR study by Hornemanna et al., [Hornemanna, Korthb, Oeschb, Rieka, Widera, Wuethricha, Glockshubera, Recombinant full-length murine prion protein, mPrP (23-231): purification and spectroscopic characterization, FEBS Lett. 413 (1997) 277-281] has indicated that N96, D143, and T189 in moPrP are localized in a Cu 2+ binding region, Helix1 and Helix2, respectively, three recombinant moPrP mutations (N96C, D143C, and T189C) were expressed in an Escherichia coli system, and then refolded by dialysis under low pH and purified by reverse-phase HPLC. By using the preparation, we succeeded in preserving a target cystein residue without alteration of the α-helix structure of moPrP and were able to apply SDSL-ESR with a methane thiosulfonate spin label to the full-length prion protein. The rotational correlation times (τ) of 1.1, 3.3, and 4.8 ns were evaluated from the X-band ESR spectra at pH 7.4 and 20 deg C for N96R1, D143R1, and T189R1, respectively. τ reflects the fact that the Cu 2+ binding region is more flexible than Helix1 or Helix2. ESR spectra recorded at various temperatures revealed two phases together with a transition point at around 20 deg C in D143R1 and T189R1, but not in N96R1. With the variation of pH from 4.0 to 7.8, ESR spectra of T189R1 at 20 deg C showed a gradual increase of τ from 2.9 to 4.8 ns. On the other hand, the pH-dependent conformational changes in N96R1 and D143R1 were negligible. These results indicated that T189 located in Helix2 possessed a structure sensitive to physiological pH changes; simultaneously, N96 in the Cu 2+ binding region and D143 in Helix1 were conserved

  13. Nevada Test Site-Directed Research and Development, FY 2007 Report

    International Nuclear Information System (INIS)

    Wil Lewis, editor

    2008-01-01

    The Nevada Test Site-Directed Research and Development (SDRD) program completed a very successful year of research and development activities in FY 2007. Twenty-nine new projects were selected for funding this year, and eight projects started in FY 2006 were brought to conclusion. The total funds expended by the SDRD program were $5.67 million, for an average per-project cost of $153 thousand. An external audit conducted in September 2007 verified that appropriate accounting practices were applied to the SDRD program. Highlights for the year included: programmatic adoption of 8 SDRD-developed technologies; the filing of 9 invention disclosures for innovation evolving from SDRD projects; participation in the tri-Lab Laboratory Directed Research and Development (LDRD) and SDRD Symposium that was broadly attended by Nevada Test Site (NTS), National Nuclear Security Administration (NNSA), LDRD, U.S. Department of Homeland Security (DHS), and U.S. Department of Defense (DoD) representatives; peer reviews of all FY 2007 projects; and the successful completion of 37 R and D projects, as presented in this report. In response to a company-wide call, authors throughout the NTS complex submitted 182 proposals for FY 2007 SDRD projects. The SDRD program has seen a dramatic increase in the yearly total of submitted proposals--from 69 in FY 2002 to 182 this year--while the number of projects funded has actually decreased from a program high of 57 in FY 2004. The overall effect of this trend has helped ensure an increasingly competitive program that benefited from a broader set of innovative ideas, making project selection both challenging and rewarding. Proposals were evaluated for technical merit, including such factors as innovation, probability of success, potential benefit, and mission applicability. Authors and reviewers benefited from the use of a shortfalls list entitled the 'NTS Technology Needs Assessment' that was compiled from NTS, National Weapons Laboratory (NWL

  14. Molecular modeling of human MT2 melatonin receptor: the role of Val204, Leu272 and Tyr298 in ligand binding

    Czech Academy of Sciences Publication Activity Database

    Mazna, Petr; Obšilová, Veronika; Jelínková, Irena; Balík, Aleš; Berka, K.; Sovová, Žofie; Ettrich, Rüdiger; Svoboda, Petr; Obšil, T.; Teisinger, Jan

    2004-01-01

    Roč. 91, č. 4 (2004), s. 836-842 ISSN 0022-3042 R&D Projects: GA ČR GA309/02/1479; GA ČR GA309/04/0496; GA ČR GA204/03/0714; GA AV ČR IAA5011103; GA AV ČR IAA5011408; GA AV ČR KJB5011308; GA MŠk LN00A141 Institutional research plan: CEZ:AV0Z5011922; CEZ:MSM 113100001 Keywords : homology modeling * MT2 melatonin receptor * site-directed mutagenesis Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 4.824, year: 2004

  15. Radiation mutagenesis in selection of apple trees

    International Nuclear Information System (INIS)

    Kolontaev, V.M.; Kolontaev, Yu.V.

    1977-01-01

    After X-radiation of grafts of antonovka apple trees, three groups of morphological mutants, namely, weak-, average- and violently-growing, have been revealed. Although the mutation spectrum has some indefinite character a dose of 6 kR causes, more frequently and in a greater number, the weak-growing mutants, and a dose of 2 kR, the violently-growing ones. Mutants of each group differ in the precociousness (precocious and latefruiting), type of fruiting (nospur and spur) and yield (high- and low-yielding). Using the method of radiation mutagenesis it is possible to rise the frequency and spectrum of somatic mutability of antonovka apple trees and to induce forms having valuable features

  16. Scoring function to predict solubility mutagenesis

    Directory of Open Access Journals (Sweden)

    Deutsch Christopher

    2010-10-01

    Full Text Available Abstract 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.

  17. Site-directed zebrafish transgenesis into single landing sites with the phiC31 integrase system.

    Science.gov (United States)

    Mosimann, Christian; Puller, Ann-Christin; Lawson, Katy L; Tschopp, Patrick; Amsterdam, Adam; Zon, Leonard I

    2013-08-01

    Linear DNA-based and Tol2-mediated transgenesis are powerful tools for the generation of transgenic zebrafish. However, the integration of multiple copies or transgenes at random genomic locations complicates comparative transgene analysis and makes long-term transgene stability unpredictable with variable expression. Targeted, site-directed transgene integration into pre-determined genomic loci can circumvent these issues. The phiC31 integrase catalyzes the unidirectional recombination reaction between heterotypic attP and attB sites and is an efficient platform for site-directed transgenesis. We report the implementation of the phiC31 integrase-mediated attP/attB recombination for site-directed zebrafish transgenics of attB-containing transgene vectors into single genomic attP landing sites. We generated Tol2-based single-insertion attP transgenic lines and established their performance in phiC31 integrase-catalyzed integration of an attB-containing transgene vector. We found stable germline transmission into the next generation of an attB reporter transgene in 34% of all tested animals. We further characterized two functional attP landing site lines and determined their genomic location. Our experiments also demonstrate tissue-specific transgene applications as well as long-term stability of phiC31-mediated transgenes. Our results establish phiC31 integrase-controlled site-directed transgenesis into single, genomic attP sites as space-, time-, and labor-efficient zebrafish transgenesis technique. The described reagents are available for distribution to the zebrafish community. Copyright © 2013 Wiley Periodicals, Inc.

  18. C-to-U editing and site-directed RNA editing for the correction of genetic mutations.

    Science.gov (United States)

    Vu, Luyen Thi; Tsukahara, Toshifumi

    2017-07-24

    Cytidine to uridine (C-to-U) editing is one type of substitutional RNA editing. It occurs in both mammals and plants. The molecular mechanism of C-to-U editing involves the hydrolytic deamination of a cytosine to a uracil base. C-to-U editing is mediated by RNA-specific cytidine deaminases and several complementation factors, which have not been completely identified. Here, we review recent findings related to the regulation and enzymatic basis of C-to-U RNA editing. More importantly, when C-to-U editing occurs in coding regions, it has the power to reprogram genetic information on the RNA level, therefore it has great potential for applications in transcript repair (diseases related to thymidine to cytidine (T>C) or adenosine to guanosine (A>G) point mutations). If it is possible to manipulate or mimic C-to-U editing, T>C or A>G genetic mutation-related diseases could be treated. Enzymatic and non-enzymatic site-directed RNA editing are two different approaches for mimicking C-to-U editing. For enzymatic site-directed RNA editing, C-to-U editing has not yet been successfully performed, and in theory, adenosine to inosine (A-to-I) editing involves the same strategy as C-to-U editing. Therefore, in this review, for applications in transcript repair, we will provide a detailed overview of enzymatic site-directed RNA editing, with a focus on A-to-I editing and non-enzymatic site-directed C-to-U editing.

  19. Evaluation of the catalytic mechanism of AICAR transformylase by pH-dependent kinetics, mutagenesis, and quantum chemical calculations.

    Science.gov (United States)

    Shim, J H; Wall, M; Benkovic, S J; Díaz, N; Suárez, D; Merz, K M

    2001-05-23

    The catalytic mechanism of 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase) is evaluated with pH dependent kinetics, site-directed mutagenesis, and quantum chemical calculations. The chemistry step, represented by the burst rates, was not pH-dependent, which is consistent with our proposed mechanism that the 4-carboxamide of AICAR assists proton shuttling. Quantum chemical calculations on a model system of 5-amino-4-carboxamide imidazole (AICA) and formamide using the B3LYP/6-31G level of theory confirmed that the 4-carboxamide participated in the proton-shuttling mechanism. The result also indicated that the amide-assisted mechanism is concerted such that the proton transfers from the 5-amino group to the formamide are simultaneous with nucleophilic attack by the 5-amino group. Because the process does not lead to a kinetically stable intermediate, the intramolecular proton transfer from the 5-amino group through the 4-carboxamide to the formamide proceeds in the same transition state. Interestingly, the calculations predicted that protonation of the N3 of the imidazole of AICA would reduce the energy barrier significantly. However, the pK(a) of the imidazole of AICAR was determined to be 3.23 +/- 0.01 by NMR titration, and AICAR is likely to bind to the enzyme with its imidazole in the free base form. An alternative pathway was suggested by modeling Lys266 to have a hydrogen-bonding interaction with the N3 of the imidazole of AICAR. Lys266 has been implicated in catalysis based on mutagenesis studies and the recent X-ray structure of AICAR Tfase. The quantum chemical calculations on a model system that contains AICA complexed with CH3NH3+ as a mimic of the Lys residue confirmed that such an interaction lowered the activation energy of the reaction and likewise implicated the 4-carboxamide. To experimentally verify this hypothesis, we prepared the K266R mutant and found that its kcat is reduced by 150-fold from that of the wild type

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  1. Structure-Based and Random Mutagenesis Approaches Increase the Organophosphate-Degrading Activity of a Phosphotriesterase Homologue from Deinococcus radiodurans

    Energy Technology Data Exchange (ETDEWEB)

    Hawwa, Renda; Larsen, Sonia D.; Ratia, Kiira; Mesecar, Andrew D.; (UIC)

    2010-11-09

    An enzyme from the amidohydrolase family from Deinococcus radiodurans (Dr-OPH) with homology to phosphotriesterase has been shown to exhibit activity against both organophosphate (OP) and lactone compounds. We have characterized the physical properties of Dr-OPH and have found it to be a highly thermostable enzyme, remaining active after 3 h of incubation at 60 C and withstanding incubation at temperatures up to 70 C. In addition, it can withstand concentrations of at least 200 mg/mL. These properties make Dr-OPH a promising candidate for development in commercial applications. However, compared to the most widely studied OP-degrading enzyme, that from Pseudomonas diminuta, Dr-OPH has low hydrolytic activity against certain OP substrates. Therefore, we sought to improve the OP-degrading activity of Dr-OPH, specifically toward the pesticides ethyl and methyl paraoxon, using structure-based and random approaches. Site-directed mutagenesis, random mutagenesis, and site-saturation mutagenesis were utilized to increase the OP-degrading activity of Dr-OPH. Out of a screen of more than 30,000 potential mutants, a total of 26 mutant enzymes were purified and characterized kinetically. Crystal structures of w.t. Dr-OPH, of Dr-OPH in complex with a product analog, and of 7 mutant enzymes were determined to resolutions between 1.7 and 2.4 {angstrom}. Information from these structures directed the design and production of 4 additional mutants for analysis. In total, our mutagenesis efforts improved the catalytic activity of Dr-OPH toward ethyl and methyl paraoxon by 126- and 322-fold and raised the specificity for these two substrates by 557- and 183-fold, respectively. Our work highlights the importance of an iterative approach to mutagenesis, proving that large rate enhancements are achieved when mutations are made in already active mutants. In addition, the relationship between the kinetic parameters and the introduced mutations has allowed us to hypothesize on those

  2. Effective mutagenesis of Arabidopsis by heavy ion beam-irradiation

    International Nuclear Information System (INIS)

    Yamamoto, Y.Y.; Saito, H.; Ryuto, H.; Fukunishi, N.; Yoshida, S.; Abe, T.

    2005-01-01

    Full text: Arabidopsis researches frequently include the genetic approach, so efficient, convenient, and safe methods for mutagenesis are required. Currently, the most popular method for in house mutagenesis is application of EMS. Although this method is very effective, its base substitution-type mutations often gives leaky mutants with residual gene functions, leading some difficulty in understanding the corresponding gene functions. Heavy ion beam generated by accelerators gives highest energy transfer rates among known radiation-based mutagenesis methods including X ray, gamma ray, fast neutron, electron and proton irradiation. This feature is thought to give high frequency of the double strand break of genomic DNA and resultant short deletions, resulting frame shift-type mutations. At RIKEN Accelerator Research Facility (RARF, http://www.rarf.riken.go.jp/index-e.html), we have optimized conditions for effective mutagenesis of Arabidopsis regarding to ion species and irradiation dose, and achieved comparable mutation rates to the method with EMS. (author)

  3. Symposium on molecular and cellular mechanisms of mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    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)

  4. Symposium on molecular and cellular mechanisms of mutagenesis

    International Nuclear Information System (INIS)

    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

  5. Isotope effects for deuterium transfer and mutagenesis of Tyr187 provide insight into controlled radical chemistry in adenosylcobalamin-dependent ornithine 4,5-aminomutase.

    Science.gov (United States)

    Makins, Caitlyn; Whitelaw, Doug A; Mu, Changhua; Walsby, Charles J; Wolthers, Kirsten R

    2014-08-26

    Adenosylcobalamin-dependent ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii utilizes pyridoxal 5'-phosphate (PLP) to interconvert d-ornithine to 2,4-diaminopentanoate via a multistep mechanism that involves two hydrogen transfer steps. Herein, we uncover features of the OAM catalytic mechanism that differentiate it from its homologue, the more catalytically promiscuous lysine 5,6-aminomutase. Kinetic isotope effects (KIEs) with dl-ornithine-3,3,4,4,5,5-d6 revealed a diminished (D)kcat/Km of 2.5 ± 0.4 relative to a (D)kcat of 7.6 ± 0.5, suggesting slow release of the substrate from the active site. In contrast, a KIE was not observed on the rate constant associated with Co-C bond homolysis as this step is likely "gated" by the formation of the external aldimine. The role of tyrosine 187, which lies planar to the PLP pyridine ring, was also investigated via site-directed mutagenesis. The 25- and 1260-fold reduced kcat values for Y187F and Y187A, respectively, are attributed to a slower rate of external aldimine formation and a diminution of adenosylcobalamin Co-C bond homolysis. Notably, electron paramagnetic resonance studies of Y187F suggest that the integrity of the active site is maintained as cob(II)alamin and the PLP organic radical (even at lower concentrations) remain tightly exchange-coupled. Modeling of d-lysine and l-β-lysine into the 5,6-LAM active site reveals interactions between the substrate and protein are weaker than those in OAM and fewer in number. The combined data suggest that the level of protein-substrate interactions in aminomutases not only influences substrate specificity, but also controls radical chemistry.

  6. Structure-function analysis of porcine cytochrome P450 3A29 in the hydroxylation of T-2 toxin as revealed by docking and mutagenesis studies.

    Directory of Open Access Journals (Sweden)

    Guyue Cheng

    Full Text Available T-2 toxin, one of the type A trichothecenes, presents a potential hazard to human and animal health. Our previous work demonstrated that porcine cytochrome P450 3A29 (CYP3A29 played an important role in the hydroxylation of T-2 toxin. To identify amino acids involved in this metabolic process, T-2 toxin was docked into a homology model of CYP3A29 based on a crystal structure of CYP3A4 using AutoDock 4.0. Nine residues of CYP3A29, Arg105, Arg106, Phe108, Ser119, Lys212, Phe213, Phe215, Arg372 and Glu374, which were found within 5 Å around T-2 toxin were subjected to site-directed mutagenesis. In the oxidation of nifedipine, the CLint value of R106A was increased by nearly two-folds compared with the wild-type CYP3A29, while the substrate affinities and CLint values of S119A and K212A were significantly reduced. In the hydroxylation of T-2 toxin, the generation of 3'-OH-T-2 by R105A, S119A and K212A was significantly less than that by the wild-type, whereas R106A slightly increased the generation of 3'-OH-T-2. These results were further confirmed by isothermal titration calorimetry analysis, suggesting that these four residues are important in the hydroxylation of T-2 toxin and Arg105 may be a specific recognition site for the toxin. Our study suggests a possible structure-function relationship of CYP3A29 in the hydroxylation of T-2 toxin, providing with new insights into the mechanism of CYP3A enzymes in the biotransformation of T-2 toxin.

  7. Yeasts acquire resistance secondary to antifungal drug treatment by adaptive mutagenesis.

    Directory of Open Access Journals (Sweden)

    David Quinto-Alemany

    Full Text Available Acquisition of resistance secondary to treatment both by microorganisms and by tumor cells is a major public health concern. Several species of bacteria acquire resistance to various antibiotics through stress-induced responses that have an adaptive mutagenesis effect. So far, adaptive mutagenesis in yeast has only been described when the stress is nutrient deprivation. Here, we hypothesized that adaptive mutagenesis in yeast (Saccharomyces cerevisiae and Candida albicans as model organisms would also take place in response to antifungal agents (5-fluorocytosine or flucytosine, 5-FC, and caspofungin, CSP, giving rise to resistance secondary to treatment with these agents. We have developed a clinically relevant model where both yeasts acquire resistance when exposed to these agents. Stressful lifestyle associated mutation (SLAM experiments show that the adaptive mutation frequencies are 20 (S. cerevisiae -5-FC, 600 (C. albicans -5-FC or 1000 (S. cerevisiae--CSP fold higher than the spontaneous mutation frequency, the experimental data for C. albicans -5-FC being in agreement with the clinical data of acquisition of resistance secondary to treatment. The spectrum of mutations in the S. cerevisiae -5-FC model differs between spontaneous and acquired, indicating that the molecular mechanisms that generate them are different. Remarkably, in the acquired mutations, an ectopic intrachromosomal recombination with an 87% homologous gene takes place with a high frequency. In conclusion, we present here a clinically relevant adaptive mutation model that fulfils the conditions reported previously.

  8. Active site-directed alkylation of Na+-K+-ATPase by digitalis sulphonate derivatives of different lipophilicity.

    Science.gov (United States)

    Fricke, U.; Klaus, W.; Rogatti, M.

    1981-01-01

    1 Sulphonate derivatives of k-strophanthidin and digitoxigenin were tested as active site-directed labels of Na+-K+-adenosine triphosphatase (Na+-ATPase) from guinea-pig heart. 2 Lipophilicity ranged between P = 93 for strophanthidin-3-tosyloxy-acetate (STA) and P = 3028 for digitoxigenin-3-tosyloxy-acetate (DTA). 3 Although the alkylating moiety of STA and DTA was identical, the reversibility of Na+-K+-ATPase inhibition varied appreciably (82% and 35% respectively). 4 It is concluded that lipophilicity contributes considerably to the irreversible binding of alkylating cardiotonic steroids to myocardial Na+-K+-ATPase. PMID:6261865

  9. The Roles of UmuD in Regulating Mutagenesis

    Directory of Open Access Journals (Sweden)

    Jaylene N. Ollivierre

    2010-01-01

    Full Text Available All organisms are subject to DNA damage from both endogenous and environmental sources. DNA damage that is not fully repaired can lead to mutations. Mutagenesis is now understood to be an active process, in part facilitated by lower-fidelity DNA polymerases that replicate DNA in an error-prone manner. Y-family DNA polymerases, found throughout all domains of life, are characterized by their lower fidelity on undamaged DNA and their specialized ability to copy damaged DNA. Two E. coli Y-family DNA polymerases are responsible for copying damaged DNA as well as for mutagenesis. These DNA polymerases interact with different forms of UmuD, a dynamic protein that regulates mutagenesis. The UmuD gene products, regulated by the SOS response, exist in two principal forms: UmuD2, which prevents mutagenesis, and UmuD2′, which facilitates UV-induced mutagenesis. This paper focuses on the multiple conformations of the UmuD gene products and how their protein interactions regulate mutagenesis.

  10. The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

    International Nuclear Information System (INIS)

    Hoskins, J.T.; Zhou, Z.; Harding, P.A.

    2008-01-01

    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 108/121 , HB-EGF-Cys/Ser 116/132 , and HB-EGF-Cys/Ser 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 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 108/121 and HB-EGF-Cys/Ser 116/132 stable cell lines which exhibited accumulated expression immediately outside the nucleus. rHB-EGF, HB-EGF, and HB-EGF 134/143 proteins competed with 125 I-EGF in an A431 competitive binding assay, whereas HB-EGF-Cys/Ser 108/121 and HB-EGF-Cys/Ser 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 134/143 antagonizes EGFRs

  11. Mutagenesis and functional analysis of the pore-forming toxin HALT-1 from Hydra magnipapillata.

    Science.gov (United States)

    Liew, Yvonne Jing Mei; Soh, Wai Tuck; Jiemy, William Febry; Hwang, Jung Shan

    2015-02-03

    Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1-4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding.

  12. Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata

    Directory of Open Access Journals (Sweden)

    Yvonne Jing Mei Liew

    2015-02-01

    Full Text Available Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1 has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1–4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding.

  13. Random mutagenesis of 1-aminocyclopropane-1-carboxylate synthase: a key enzyme in ethylene biosynthesis.

    Science.gov (United States)

    Tarun, A S; Lee, J S; Theologis, A

    1998-08-18

    1-Aminocyclopropane-1-carboxylate synthase (ACC synthase, EC 4.4.1. 14) catalyzes the rate-limiting step in the ethylene biosynthetic pathway in plants. To determine the amino acid residues critical for the structure and function of this enzyme, the tomato Le-ACS2 isoenzyme has been subjected to both site-directed and PCR random mutagenesis. Mutant ACC synthases with reduced enzyme activity have been selected by using a genetic screen based on the functional complementation of an Escherichia coli Ile auxotroph that has been engineered to express ACC deaminase from Pseudomonas sp. The DNA sequence of almost 1,000 clones has been determined, and 334 single missense mutations have been selected for analysis. We have identified three classes of mutants based on their activity and expression in E. coli. Class I and II mutants have the same level of protein expression as the wild type, but their enzyme activity is reduced to 0-5% and 5-50%, respectively. Class III mutants have neither activity nor detectable protein expression. The inactive mutations are clustered in regions that are highly conserved among various ACC synthases. This library of mutants will facilitate the elucidation of structure-function relationships of this regulatory enzyme.

  14. Site‐directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of Rab7b paralogue in the model eukaryote Paramecium octaurelia

    Directory of Open Access Journals (Sweden)

    E. Wyroba

    2016-04-01

    Full Text Available Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. The role of post‐translational modifications (PTM in proper targeting of Paramecium Rab7b paralogue – distinct from that of Rab7a directly involved in phagocytosis ‐ was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala200 resulted in diminished incorporation of [P32] by 37.4% and of 32 [C14–]UDP‐glucose by 24%, respectively, into recombinant Rab7b_200 in comparison to the non‐mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells contrary to non‐ mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.63+ characteristic for the glycan group attached to Thr200 in Rab7b using nano LC‐MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P32]and  [C14‐]UDP‐ glucose, the suggested composition of the adduct attached to Thr200 might be (Hex1(HexNAc1(Phos3 or (HexNAc1 (Deoxyhexose1 (Phos1 (HexA1. These data indicate that PTM of Thr200 located in the hypervariable C‐region of Rab7b in Paramecium is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a.   

  15. Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans

    International Nuclear Information System (INIS)

    Prasannan, Priya; Suliman, Huda S.; Robertus, Jon D.

    2009-01-01

    Fungal methionine synthase catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine to create methionine. The enzyme, called Met6p in fungi, is required for the growth of the pathogen Candida albicans, and is consequently a reasonable target for antifungal drug design. In order to understand the mechanism of this class of enzyme, we created a three-dimensional model of the C. albicans enzyme based on the known structure of the homologous enzyme from Arabidopsis thaliana. A fusion protein was created and shown to have enzyme activity similar to the wild-type Met6p. Fusion proteins containing mutations at eight key sites were expressed and assayed in this background. The D614 carboxylate appears to ion pair with the amino group of homocysteine and is essential for activity. Similarly, D504 appears to bind to the polar edge of the folate and is also required for activity. Other groups tested have lesser roles in substrate binding and catalysis.

  16. Kinetic analysis of site-directed mutants of methionine synthase from Candida albicans

    Energy Technology Data Exchange (ETDEWEB)

    Prasannan, Priya; Suliman, Huda S. [Institute of Cellular and Molecular Biology, Department of Chemistry and Biochemistry, 1 University Station A5300, University of Texas, Austin, TX 78712 (United States); Robertus, Jon D., E-mail: jrobertus@mail.utexas.edu [Institute of Cellular and Molecular Biology, Department of Chemistry and Biochemistry, 1 University Station A5300, University of Texas, Austin, TX 78712 (United States)

    2009-05-15

    Fungal methionine synthase catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine to create methionine. The enzyme, called Met6p in fungi, is required for the growth of the pathogen Candida albicans, and is consequently a reasonable target for antifungal drug design. In order to understand the mechanism of this class of enzyme, we created a three-dimensional model of the C. albicans enzyme based on the known structure of the homologous enzyme from Arabidopsis thaliana. A fusion protein was created and shown to have enzyme activity similar to the wild-type Met6p. Fusion proteins containing mutations at eight key sites were expressed and assayed in this background. The D614 carboxylate appears to ion pair with the amino group of homocysteine and is essential for activity. Similarly, D504 appears to bind to the polar edge of the folate and is also required for activity. Other groups tested have lesser roles in substrate binding and catalysis.

  17. PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. II. Characterization Of the binding and cleavage abilities by site-directed mutagenesis.

    OpenAIRE

    Komori, K; Ichiyanagi, K; Morikawa, K; Ishino, Y

    1999-01-01

    PI- Pfu I and PI- Pfu II from Pyrococcus furiosus are homing endonucleases, as shown in the accompanying paper. These two endonucleases are produced by protein splicing from the precursor protein including ribonucleotide reductase (RNR). We show here that both enzymes specifically interact with their substrate DNA and distort the DNA strands by 73 degrees and 67 degrees, respectively. They have two copies of the amino acid sequence motif LAGLIDADG, which is present in the majority of homing e...

  18. Species-specific differences of the spectroscopic properties of P700 - Analysis of the influence of non-conserved amino acid residues by site-directed mutagenesis of photosystem I from Chlamydomonas reinhardtii

    NARCIS (Netherlands)

    Witt, H.; Bordignon, E.; Carbonera, D.; Dekker, J.P.; Karapetyan, N.; Teutloff, C.; Webber, A.; Lubitz, W.; Schlodder, E.

    2003-01-01

    We applied optical spectroscopy, magnetic resonance techniques, and redox titrations to investigate the properties of the primary electron donor P700 in photosystem I (PS I) core complexes from cyanobacteria (Thermosynechococcus elongatus, Spirulina platensis, and Synechocystis sp. PCC 6803), algae

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

    Directory of Open Access Journals (Sweden)

    Grégory Caignard

    2014-09-01

    Full Text Available 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.

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

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    Takashi Nakanishi

    Full Text Available 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.

  1. Site directed recombination

    Science.gov (United States)

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  2. Elucidating the design principles of photosynthetic electron-transfer proteins by site-directed spin labeling EPR spectroscopy.

    Science.gov (United States)

    Ishara Silva, K; Jagannathan, Bharat; Golbeck, John H; Lakshmi, K V

    2016-05-01

    Site-directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy is a powerful tool to determine solvent accessibility, side-chain dynamics, and inter-spin distances at specific sites in biological macromolecules. This information provides important insights into the structure and dynamics of both natural and designed proteins and protein complexes. Here, we discuss the application of SDSL EPR spectroscopy in probing the charge-transfer cofactors in photosynthetic reaction centers (RC) such as photosystem I (PSI) and the bacterial reaction center (bRC). Photosynthetic RCs are large multi-subunit proteins (molecular weight≥300 kDa) that perform light-driven charge transfer reactions in photosynthesis. These reactions are carried out by cofactors that are paramagnetic in one of their oxidation states. This renders the RCs unsuitable for conventional nuclear magnetic resonance spectroscopy investigations. However, the presence of native paramagnetic centers and the ability to covalently attach site-directed spin labels in RCs makes them ideally suited for the application of SDSL EPR spectroscopy. The paramagnetic centers serve as probes of conformational changes, dynamics of subunit assembly, and the relative motion of cofactors and peptide subunits. In this review, we describe novel applications of SDSL EPR spectroscopy for elucidating the effects of local structure and dynamics on the electron-transfer cofactors of photosynthetic RCs. Because SDSL EPR Spectroscopy is uniquely suited to provide dynamic information on protein motion, it is a particularly useful method in the engineering and analysis of designed electron transfer proteins and protein networks. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. Copyright © 2016. Published by Elsevier B.V.

  3. In vitro Inactivation of Latent HSV by Targeted Mutagenesis Using an HSV-specific Homing Endonuclease

    Directory of Open Access Journals (Sweden)

    Martine Aubert

    2014-01-01

    Full Text Available Following acute infection, herpes simplex virus (HSV establishes latency in sensory neurons, from which it can reactivate and cause recurrent disease. Available antiviral therapies do not affect latent viral genomes; therefore, they do not prevent reactivation following therapy cessation. One possible curative approach involves the introduction of DNA double strand breaks in latent HSV genomes by rare-cutting endonucleases, leading to mutagenesis of essential viral genes. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Coexpression of the 3′-exonuclease Trex2 with HEs increased HE-mediated mutagenesis frequencies up to sixfold. Following HSV1m5/Trex2 delivery with adeno-associated viral (AAV vectors, the target site was mutated in latent HSV genomes with no detectable cell toxicity. Importantly, HSV production by latently infected cells after reactivation was decreased after HSV1m5/Trex2 exposure. Exposure to histone deacetylase inhibitors prior to HSV1m5/Trex2 treatment increased mutagenesis frequencies of latent HSV genomes another two- to fivefold, suggesting that chromatin modification may be a useful adjunct to gene-targeting approaches. These results support the continuing development of HEs and other nucleases (ZFNs, TALENs, CRISPRs for cure of chronic viral infections.

  4. Genetic modifications of established varieties of potato through mutagenesis

    International Nuclear Information System (INIS)

    Brown, C.R.

    1984-01-01

    Owing to the high intercrossability of improved clones with primitive cultivars and many wild species there is little justification for use of induced mutations in potato to increase variability per se. Modification of certain traits while leaving the genotype basically intact is a promising use of mutagenesis in potato. The successful curing of defects in clones will depend on the establishment a priori of three principles. First, the clones undergoing mutagenesis should be well established varieties tolerant or resistant to the major biotic and abiotic stresses in the area of cultivation. The yield and culinary quality should also be considered high. Second, there should exist some indication that the variation desired is induceable, either through reports of natural intra-clone variation or previous mutagenesis studies. Third, initial screening should be done in virus-free materials

  5. Genetic and physiological factors affecting repair and mutagenesis in yeast

    International Nuclear Information System (INIS)

    Lemontt, J.F.

    1979-01-01

    Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data, and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described particularly in relation to their involvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus, are shown to also play a role in repair and mutagenesis

  6. Genetic and physiological factors affecting repair and mutagenesis in yeast

    International Nuclear Information System (INIS)

    Lemontt, J.F.

    1979-01-01

    Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described, particularly in relation to their imvolvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus are shown to also play a role in repair and mutagenesis

  7. Genetic and physiological factors affecting repair and mutagenesis in yeast

    Energy Technology Data Exchange (ETDEWEB)

    Lemontt, J F

    1979-01-01

    Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data, and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described particularly in relation to their involvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus, are shown to also play a role in repair and mutagenesis.

  8. Genetic and physiological factors affecting repair and mutagenesis in yeast

    Energy Technology Data Exchange (ETDEWEB)

    Lemontt, J F

    1979-01-01

    Current views of DNA repair and mutagenesis in the yeast Saccharomyces cerevisiae are discussed in the light of recent data and with emphasis on the isolation and characterization of genetically well-defined mutations that affect DNA metabolism in general (including replication and recombination). Various pathways of repair are described, particularly in relation to their imvolvement in mutagenic mechanisms. In addition to genetic control, certain physiological factors such as cell age, DNA replication, and the regulatory state of the mating-type locus are shown to also play a role in repair and mutagenesis.

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

    DEFF Research Database (Denmark)

    Nilsson, Martin; Christiansen, Natalia; Høiby, Niels

    2014-01-01

    ATs-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...

  10. The Site-Directed A184S Mutation in the HTH Domain of the Global Regulator IrrE Enhances Deinococcus radiodurans R1 Tolerance to UV Radiation and MMC Shock.

    Science.gov (United States)

    Zhang, Chen; Zhou, Zhengfu; Zhang, Wei; Chen, Zhen; Song, Yuan; Lu, Wei; Lin, Min; Chen, Ming

    2015-12-28

    IrrE is a highly conserved global regulator in the Deinococcus genus and contributes to survival from high doses of UV radiation, ionizing radiation, and desiccation. Drad-IrrE and Dgob-IrrE from Deinococcus radiodurans and Deinococcus gobiensis I-0 each share 66% sequence identity. However, Dgob-IrrE showed a stronger protection phenotype against UV radiation than Drad- IrrE in the D. radiodurans irrE-deletion mutant (ΔirrE), which may be due to amino acid residues differences around the DNA-binding HTH domain. Site-directed mutagenesis was used to generate a Drad-IrrE A184S single mutant, which has been characterized and compared with the ΔirrE mutant complemented strain with Drad-irrE, designated ΔirrE-E. The effects of the A184S mutation following UV radiation and mitomycin C (MMC) shock were determined. The A184S mutant displayed significantly increased resistance to UV radiation and MMC shock. The corresponding A184 site in Dgob-IrrE was inversely mutated, generating the S131A mutant, which exhibited a loss of resistance against UV radiation, MMC shock, and desiccation. qPCR analysis revealed that critical genes in the DNA repair system, such as recA, pprA, uvrA, and ddrB, were remarkably induced after UV radiation and MMC shock in the ΔirrE-IE and A184S mutants. These data suggested that A184S improves the ability against UV radiation and MMC shock, providing new insights into the modification of IrrE. We speculated that the serine residue may determine the efficiency of DNA binding, leading to the increased expression of IrrE-dependent genes important for protection against DNA damage.

  11. Topology of OxlT, the oxalate transporter of Oxalobacter formigenes, determined by site-directed fluorescence labeling.

    Science.gov (United States)

    Ye, L; Jia, Z; Jung, T; Maloney, P C

    2001-04-01

    The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 single-cysteine variants, each generated in a His(9)-tagged cysteine-less host. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material purified by Ni2+ linked affinity chromatography. A position was assigned an external location if its single-cysteine derivative reacted with OGM added to intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not be assigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assignment. Our findings document a simple and rapid method for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis.

  12. Methods for targetted mutagenesis in gram-positive bacteria

    Science.gov (United States)

    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.

  13. Effect of Colchicine Induced Mutagenesis on Growth and Yield of ...

    African Journals Online (AJOL)

    Chemical mutagenesis through the use of colchicine on the seeds of two varieties of sesame (Sesamum indicum L. Var. Ex-Sudan and E-8) with the aim of inducing variability that could be exploited in the genetic improvement of its growth and yield was carried out. The sesame seeds were treated with colchicines at four ...

  14. Targeted mutagenesis using CRISPR/Cas in inbred potatoes

    Science.gov (United States)

    Targeted mutagenesis using sequence-specific nucleases (SSNs) has been well established in several important crop species, but is in need of improvement in potato (Solanum tuberosum L.). For over a century, potatoes have been bred as autotetraploids (2n = 4x = 48), relying on F1 selections and clona...

  15. The European dimension for the mouse genome mutagenesis

    Czech Academy of Sciences Publication Activity Database

    Auwerx, J.; Avner, P.; Baldock, R.; Ballabio, A.; Balling, R.; Barbacid, M.; Berns, A.; Bradley, A.; Brown, S.; Carmeliet, P.; Chambon, P.; Cox, R.; Davidson, D.; Davies, K.; Duboule, D.; Forejt, Jiří; Granucci, F.; Hastie, N.; Angelis, M. H. de; Jackson, I.; Kioussis, D.; Kollias, G.; Lathrop, M.; Lendahl, U.; Malumbres, M.; von Melchner, H.; Müller, W.; Partanen, J.; Ricciardi-Castagnoli, P.; Rigby, P.; Rosen, B.; Rosenthal, N.; Skarnes, B.; Stewart, A. F.; Thornton, J.; Tocchini-Valentini, G.; Wagner, E.; Wahli, W.; Wurst, W.

    2004-01-01

    Roč. 16, - (2004), s. 925-927 ISSN 1061-4036 R&D Projects: GA MŠk(CZ) LN00A079 Institutional research plan: CEZ:AV0Z5052915 Keywords : The European Mouse Mutagenesis Consortium Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 24.695, year: 2004

  16. MtDNA mutagenesis impairs elimination of mitochondria during erythroid maturation leading to enhanced erythrocyte destruction

    NARCIS (Netherlands)

    Ahlqvist, K.J.; Leoncini, S.; Pecorelli, A.; Wortmann, S.B.; Ahola, S.; Forsstrom, S.; Guerranti, R.; Felice, C. De; Smeitink, J.; Ciccoli, L.; Hamalainen, R.H.; Suomalainen, A.

    2015-01-01

    Haematopoietic progenitor cells show special sensitivity to mitochondrial DNA (mtDNA) mutagenesis, which suggests that increased mtDNA mutagenesis could underlie anemias. Here we show that elevated mtDNA mutagenesis in mice with a proof-reading deficient mtDNA polymerase (PolG) leads to incomplete

  17. CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean.

    Science.gov (United States)

    Cai, Yupeng; Chen, Li; Liu, Xiujie; Guo, Chen; Sun, Shi; Wu, Cunxiang; Jiang, Bingjun; Han, Tianfu; Hou, Wensheng

    2018-01-01

    Flowering is an indication of the transition from vegetative growth to reproductive growth and has considerable effects on the life cycle of soya bean (Glycine max). In this study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of GmFT2a, an integrator in the photoperiod flowering pathway in soya bean. The soya bean cultivar Jack was transformed with three sgRNA/Cas9 vectors targeting different sites of endogenous GmFT2a via Agrobacterium tumefaciens-mediated transformation. Site-directed mutations were observed at all targeted sites by DNA sequencing analysis. T1-generation soya bean plants homozygous for null alleles of GmFT2a frameshift mutated by a 1-bp insertion or short deletion exhibited late flowering under natural conditions (summer) in Beijing, China (N39°58', E116°20'). We also found that the targeted mutagenesis was stably heritable in the following T2 generation, and the homozygous GmFT2a mutants exhibited late flowering under both long-day and short-day conditions. We identified some 'transgene-clean' soya bean plants that were homozygous for null alleles of endogenous GmFT2a and without any transgenic element from the T1 and T2 generations. These 'transgene-clean' mutants of GmFT2a may provide materials for more in-depth research of GmFT2a functions and the molecular mechanism of photoperiod responses in soya bean. They will also contribute to soya bean breeding and regional introduction. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  18. From classical mutagenesis to nuclease-based breeding - directing natural DNA repair for a natural end-product.

    Science.gov (United States)

    Pacher, Michael; Puchta, Holger

    2017-05-01

    Production of mutants of crop plants by the use of chemical or physical genotoxins has a long tradition. These factors induce the natural DNA repair machinery to repair damage in an error-prone way. In the case of radiation, multiple double-strand breaks (DSBs) are induced randomly in the genome, leading in very rare cases to a desirable phenotype. In recent years the use of synthetic, site-directed nucleases (SDNs) - also referred to as sequence-specific nucleases - like the CRISPR/Cas system has enabled scientists to use exactly the same naturally occurring DNA repair mechanisms for the controlled induction of genomic changes at pre-defined sites in plant genomes. As these changes are not necessarily associated with the permanent integration of foreign DNA, the obtained organisms per se cannot be regarded as genetically modified as there is no way to distinguish them from natural variants. This applies to changes induced by DSBs as well as single-strand breaks, and involves repair by non-homologous end-joining and homologous recombination. The recent development of SDN-based 'DNA-free' approaches makes mutagenesis strategies in classical breeding indistinguishable from SDN-derived targeted genome modifications, even in regard to current regulatory rules. With the advent of new SDN technologies, much faster and more precise genome editing becomes available at reasonable cost, and potentially without requiring time-consuming deregulation of newly created phenotypes. This review will focus on classical mutagenesis breeding and the application of newly developed SDNs in order to emphasize similarities in the context of the regulatory situation for genetically modified crop plants. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  19. Scaffolding functions of arrestin-2 revealed by crystal structure and mutagenesis.

    Science.gov (United States)

    Milano, Shawn K; Pace, Helen C; Kim, You-Me; Brenner, Charles; Benovic, Jeffrey L

    2002-03-12

    Arrestin binding to activated, phosphorylated G protein-coupled receptors (GPCRs) represents a critical step in regulation of light- and hormone-dependent signaling. Nonvisual arrestins, such as arrestin-2, interact with multiple proteins for the purpose of propagating and terminating signaling events. Using a combination of X-ray crystallography, molecular modeling, mutagenesis, and binding analysis, we reveal structural features of arrestin-2 that may enable simultaneous binding to phosphorylated receptor, SH3 domains, phosphoinositides, and beta-adaptin. The structure of full-length arrestin-2 thus provides a uniquely oriented scaffold for assembly of multiple, diverse molecules involved in GPCR signal transduction.

  20. Nucleic Acid-Dependent Conformational Changes in CRISPR-Cas9 Revealed by Site-Directed Spin Labeling.

    Science.gov (United States)

    Vazquez Reyes, Carolina; Tangprasertchai, Narin S; Yogesha, S D; Nguyen, Richard H; Zhang, Xiaojun; Rajan, Rakhi; Qin, Peter Z

    2017-06-01

    In a type II clustered regularly interspaced short palindromic repeats (CRISPR) system, RNAs that are encoded at the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNA and the target DNA, direct the protein into its active conformation, yet details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remain to be elucidated. Here, nucleic acid-dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) were investigated using the method of site-directed spin labeling (SDSL). Single nitroxide spin labels were attached, one at a time, at one of the two native cysteine residues (Cys80 and Cys574) of SpyCas9, and the spin-labeled proteins were shown to maintain their function. X-band continuous-wave electron paramagnetic resonance spectra of the nitroxide attached at Cys80 revealed conformational changes of SpyCas9 that are consistent with a large-scale domain re-arrangement upon binding to its RNA partner. The results demonstrate the use of SDSL to monitor conformational changes in CRISPR-Cas9, which will provide key information for understanding the mechanism of CRISPR function.

  1. The role of the bacterial mismatch repair system in SOS-induced mutagenesis: a theoretical background

    International Nuclear Information System (INIS)

    Belov, O.V.; Kapralov, M.I.; Chuluunbaatar, O.; Sweilam, N.H.

    2012-01-01

    A theoretical study is performed of the possible role of the methyl-directed mismatch repair system in the ultraviolet-induced mutagenesis of Escherichia coli bacterial cells. For this purpose, a mathematical model of the bacterial mismatch repair system is developed. Within this model, the key pathways of this type of repair are simulated on the basis of modern experimental data related to its mechanisms. Here we have modelled in detail five main pathways of DNA misincorporation removal with different DNA exonucleases. Using our calculations, we have tested the hypothesis that the bacterial mismatch repair system is responsible for the removal of the nucleotides misincorporated by DNA polymerase V (the UmuD' 2 C complex) during ultraviolet-induced SOS response. For the theoretical analysis of the mutation frequency, we have combined the proposed mathematical approach with the model of SOS-induced mutagenesis in the E.coli bacterial cell developed earlier. Our calculations support the hypothesis that methyl-directed mismatch repair influences the mutagenic effect of ultraviolet radiation

  2. Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xiaojun; Tung, Chang-Shung; Sowa, Glenna; Hatmal, Ma' mon M.; Haworth, Ian S.; Qin, Peter Z.

    2012-02-08

    The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron-Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (HT and HL) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which HT and HL stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial.

  3. Mutagenesis breeding research of Lactobacillus brevis of nitrite reduction

    Directory of Open Access Journals (Sweden)

    LI Zeli

    2015-10-01

    Full Text Available The pollution of nitrite in food became one of the focus of food safety issues,the use of biotechnology methods degrading nitrite became hotspot.The primitive strain was Lactobacillus brevis C2,preserved in our laboratory,had the ability to degrade nitrite,through composite mutagenesis of 15 W,254 nm,20 cm ultraviolet mutagenesis (UV for 120 s and 0.8% diethyl sulfate(DES in 37℃ mutation for 40 min,after screening,we successfully obtained high efficient strain of nitrite degradation,named UV6-DS2,relative to the starting strain,under the condition of 400 mg/L nitrite,after 12 h degradation,nitrite degradation rate increased from 92.8% to 97.8%,to explore its application in food was able to effectively reduce concentration of nitrite in food.

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

    Directory of Open Access Journals (Sweden)

    Kentaro Ishida

    2015-10-01

    Full Text Available 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.

  5. Targeted mutagenesis in sea urchin embryos using TALENs.

    Science.gov (United States)

    Hosoi, Sayaka; Sakuma, Tetsushi; Sakamoto, Naoaki; Yamamoto, Takashi

    2014-01-01

    Genome editing with engineered nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has been reported in various animals. We previously described ZFN-mediated targeted mutagenesis and insertion of reporter genes in sea urchin embryos. In this study, we demonstrate that TALENs can induce mutagenesis at specific genomic loci of sea urchin embryos. Injection of TALEN mRNAs targeting the HpEts transcription factor into fertilized eggs resulted in the impairment of skeletogenesis. Sequence analyses of the mutations showed that deletions and/or insertions occurred at the HpEts target site in the TALEN mRNAs-injected embryos. The results suggest that targeted gene disruption using TALENs is feasible in sea urchin embryos. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  6. Photodynamic action of methylene blue: mutagenesis and synergism

    International Nuclear Information System (INIS)

    Capella, M.A.M.

    1988-01-01

    The associated mutagenesis and the interactions with physical agents in order to potencialize its biological effects are studied. The induction of mutation in bacterias due to photodynamic action of methylene blue is presented as well as the induction of single breaks in bacterial DNA and the relationship between the repair systems, especially the SOS one. The interaction of the photodynamic therapy with low intensity electric current is discussed. (M.A.C.) [pt

  7. Environmental mutagenesis and radiation biology: The legacy of William Morgan.

    Science.gov (United States)

    Schwartz, Jeffrey L

    2017-12-01

    A symposium entitled Environmental Mutagenesis and Radiation Biology was held on September 27, 2016 to honor the memory of Dr. William F. Morgan who passed away unexpectedly on November 13, 2015. The speakers presented the latest reviews on homologous recombination repair, induced genetic instability, bystander effects, and risk estimate development. Their presentations are presented following the introduction. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9.

    Science.gov (United States)

    Liu, Yang; Merrick, Paul; Zhang, Zhengzhi; Ji, Chonghui; Yang, Bing; Fei, Shui-Zhang

    2018-02-01

    The CRISPR/Cas9 system has become a powerful tool for targeted mutagenesis. Switchgrass (Panicum virgatum L.) is a high yielding perennial grass species that has been designated as a model biomass crop by the U.S. Department of Energy. The self-infertility and high ploidy level make it difficult to study gene function or improve germplasm. To overcome these constraints, we explored the feasibility of using CRISPR/Cas9 for targeted mutagenesis in a tetraploid cultivar 'Alamo' switchgrass. We first developed a transient assay by which a non-functional green-fluorescent protein gene containing a 1-bp frameshift insertion in its 5' coding region was successfully mutated by a Cas9/sgRNA complex resulting in its restored function. Agrobacterium-mediated stable transformation of embryogenic calli derived from mature caryopses averaged a 3.0% transformation efficiency targeting the genes of teosinte branched 1(tb1)a and b and phosphoglycerate mutase (PGM). With a single construct containing two sgRNAs targeting different regions of tb1a and tb1b genes, primary transformants (T0) containing CRISPR/Cas9-induced mutations were obtained at frequencies of 95.5% (tb1a) and 11% (tb1b), respectively, with T0 mutants exhibiting increased tiller production. Meanwhile, a mutation frequency of 13.7% was obtained for the PGM gene with a CRISPR/Cas9 construct containing a single sgRNA. Among the PGM T0 mutants, six are heterozygous and one is homozygous for a 1-bp deletion in the target region with no apparent phenotypical alterations. We show that CRISPR/Cas9 system can generate targeted mutagenesis effectively and obtain targeted homozygous mutants in T0 generation in switchgrass, circumventing the need of inbreeding. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  9. Cationic antimicrobial peptides promote microbial mutagenesis and pathoadaptation in chronic infections.

    Directory of Open Access Journals (Sweden)

    Dominique H Limoli

    2014-04-01

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

  10. Theory for site-site pair distribution functions of molecular fluids. II. Approximations for the Percus--Yevick site-site direct correlation functions

    International Nuclear Information System (INIS)

    Johnson, E.

    1977-01-01

    A theory for site-site pair distribution functions of molecular fluids is derived from the Ornstein-Zernike equation. Atom-atom pair distribution functions of this theory which were obtained by using different approximations for the Percus-Yevick site-site direct correlation functions are compared

  11. Membrane docking geometry of GRP1 PH domain bound to a target lipid bilayer: an EPR site-directed spin-labeling and relaxation study.

    Directory of Open Access Journals (Sweden)

    Huai-Chun Chen

    Full Text Available The second messenger lipid PIP(3 (phosphatidylinositol-3,4,5-trisphosphate is generated by the lipid kinase PI3K (phosphoinositide-3-kinase in the inner leaflet of the plasma membrane, where it regulates a broad array of cell processes by recruiting multiple signaling proteins containing PIP(3-specific pleckstrin homology (PH domains to the membrane surface. Despite the broad importance of PIP(3-specific PH domains, the membrane docking geometry of a PH domain bound to its target PIP(3 lipid on a bilayer surface has not yet been experimentally determined. The present study employs EPR site-directed spin labeling and relaxation methods to elucidate the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP(3. The model target bilayer contains the neutral background lipid PC and both essential targeting lipids: (i PIP(3 target lipid that provides specificity and affinity, and (ii PS facilitator lipid that enhances the PIP(3 on-rate via an electrostatic search mechanism. The EPR approach measures membrane depth parameters for 18 function-retaining spin labels coupled to the PH domain, and for calibration spin labels coupled to phospholipids. The resulting depth parameters, together with the known high resolution structure of the co-complex between GRP1 PH domain and the PIP(3 headgroup, provide sufficient constraints to define an optimized, self-consistent membrane docking geometry. In this optimized geometry the PH domain engulfs the PIP(3 headgroup with minimal bilayer penetration, yielding the shallowest membrane position yet described for a lipid binding domain. This binding interaction displaces the PIP(3 headgroup from its lowest energy position and orientation in the bilayer, but the headgroup remains within its energetically accessible depth and angular ranges. Finally, the optimized docking geometry explains previous biophysical findings including mutations observed to disrupt membrane binding, and the rapid lateral

  12. AAscan, PCRdesign and MutantChecker: a suite of programs for primer design and sequence analysis for high-throughput scanning mutagenesis.

    Directory of Open Access Journals (Sweden)

    Dawei Sun

    Full Text Available Scanning mutagenesis is a powerful protein engineering technique used to study protein structure-function relationship, map binding sites and design more stable proteins or proteins with altered properties. One of the time-consuming tasks encountered in application of this technique is the design of primers for site-directed mutagenesis. Here we present an open-source multi-platform software AAscan developed to design primers for this task according to a set of empirical rules such as melting temperature, overall length, length of overlap regions, and presence of GC clamps at the 3' end, for any desired substitution. We also describe additional software tools which are used to analyse a large number of sequencing results for the presence of desired mutations, as well as related software to design primers for ligation independent cloning. We have used AAscan software to design primers to make over 700 mutants, with a success rate of over 80%. We hope that the open-source nature of our software and ready availability of freeware tools used for its development will facilitate its adaptation and further development. The software is distributed under GPLv3 licence and is available at http://www.psi.ch/lbr/aascan.

  13. Efficient and Heritable Targeted Mutagenesis in Mosses Using the CRISPR/Cas9 System.

    Science.gov (United States)

    Nomura, Toshihisa; Sakurai, Tetsuya; Osakabe, Yuriko; Osakabe, Keishi; Sakakibara, Hitoshi

    2016-12-01

    Targeted genome modification by RNA-guided nucleases derived from the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has seen rapid development in many organisms, including several plant species. In the present study, we succeeded in introducing the CRISPR/Cas9 system into the non-model organism Scopelophila cataractae, a moss that exhibits heavy metal tolerance, and the model organism Physcomitrella patens Utilizing the process by which moss plants regenerate from protoplasts, we conducted targeted mutagenesis by expression of single-chain guide RNA (sgRNA) and Cas9 in protoplasts. Using this method, the acquisition rate of strains exhibiting phenotypic changes associated with the target genes was approximately 45-69%, and strains with phenotypic changes exhibited various insertion and deletion mutations. In addition, we report that our method is capable of multiplex targeted mutagenesis (two independent genes) and also permits the efficient introduction of large deletions (∼3 kbp). These results demonstrate that the CRISPR/Cas9 system can be used to accelerate investigations of bryology and land plant evolution. © The Authors 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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 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

  15. Directed mutagenesis affects recombination in Azospirillum brasilense nif genes

    Directory of Open Access Journals (Sweden)

    C.P. Nunes

    2000-12-01

    Full Text Available In order to improve the gene transfer/mutagenesis system for Azospirillum brasilense, gene-cartridge mutagenesis was used to replace the nifD gene with the Tn5 kanamycin resistance gene. The construct was transferred to A. brasilense by electrotransformation. Of the 12 colonies isolated using the suicide plasmid pSUP202 as vector, only four did not show vector integration into the chromosome. Nevertheless, all 12 colonies were deficient in acetylene reduction, indicating an Nif- phenotype. Four Nif- mutants were analyzed by Southern blot, using six different probes spanning the nif and Km r genes and the plasmid vector. Apparently, several recombination events occurred in the mutant genomes, probably caused mainly by gene disruption owing to the mutagenesis technique used: resistance gene-cartridge mutagenesis combined with electrotransformation.Com o objetivo de melhorar os sistemas de transferência gênica e mutagênese para Azospirillum brasilense, a técnica de mutagênese através do uso de um gene marcador ("gene-cartridge mutagenesis" foi utilizada para substituir a região genômica de A. brasilense correspondente ao gene nifD por um segmento de DNA do transposon Tn5 contendo o gene que confere resistência ao antibiótico canamicina. A construção foi transferida para a linhagem de A. brasilense por eletrotransformação. Doze colônias transformantes foram isoladas com o plasmídeo suicida pSUP202 servindo como vetor. Dessas, somente quatro não possuíam o vetor integrado no cromossomo da bactéria. Independentemente da integração ou não do vetor, as 12 colônias foram deficientes na redução do gás acetileno, evidenciando o fenótipo Nif -. Quatro mutantes Nif - foram analisados através da técnica de Southern blot, utilizando-se seis diferentes fragmentos contendo genes nif, de resistência à canamicina e do vetor como sondas. Os resultados sugerem a ocorrência de eventos recombinacionais variados no genoma dos mutantes. A

  16. Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis.

    Science.gov (United States)

    Yokoyama, Keiichi; Utsumi, Hiroe; Nakamura, Takefumi; Ogaya, Daisuke; Shimba, Nobuhisa; Suzuki, Eiichiro; Taguchi, Seiichi

    2010-08-01

    Microbial transglutaminase (MTG) has been used extensively in academic research and the food industries through its cross-linking or posttranslational modification of proteins. Two enzyme engineering approaches were applied to improve MTG activity. One is a novel method of rational mutagenesis, called water-accessible surface hot-space region-oriented mutagenesis (WASH-ROM). One hundred and fifty-one point mutations were selected at 40 residues, bearing high solvent-accessibility surface area, within a 15 A space from the active site Cys64. Among them, 32 mutants showed higher specific activity than the wild type. The other is a random mutagenesis of the whole region of the MTG gene, coupled with a new plate assay screening system, using Corynebacterium Expression System CORYNEX. This in vivo system allowed us to readily distinguish the change in enzymatic activity by monitoring the intensity of enzymatic reaction-derived color zones surrounding recombinant cells. From the library of 24,000 mutants, ten were finally selected as beneficial mutants exhibiting higher specific activity than the wild type. Furthermore, we found that Ser199Ala mutant with additional N-terminal tetrapeptide showed the highest specific activity (1.7 times higher than the wild type). These various beneficial positions leading to increased specific activity of MTG were identified to achieve further enzyme improvements.

  17. UVB-induced mutagenesis in hairless λlacZ-transgenic mice

    International Nuclear Information System (INIS)

    Frijhoff, A.F.W.; Rebel, H.; Mientjes, E.J.

    1997-01-01

    UVB-induced mutagenesis was studied in hairless 40.6 transgenic mice (Muta trademark Mouse), which contain the λgt1OlacZ shuttle vector as a target for mutagenesis. Mice were exposed at the dorsal side to either single doses of 200, 500, 800, or 1000 J/m 2 UVB or to two successive irradiations of either 200 and 800 J/m 2 UVB, with intervals of 1,3, or 5 days, or to 800 and 200 J/m 2 UVB with a 5-day interval. At 23 days after the last exposure, lacZ mutant frequencies (MF) were determined in the epidermis. The lacZ MF increased linearly with increasing dose of UVB. The mutagenic effect of two successive irradiations appeared to be additive. The UV-induced mutation spectrum was dominated by G:C→A:T transitions at dipyrimidine sites. DNA-sequence analysis of spontaneously mutated phages showed a diverse spectrum consisting of insertions, deletions and G:C → A:T transitions at CpG sites. the results indicate that the hairless λlacZ-transgenic mouse is a suitable in vivo model for studying UVB-induced mutations. 29 refs., 5 tabs

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

    Energy Technology Data Exchange (ETDEWEB)

    Kermany, Mohammad [St. Jude Children' s Research Hospital; Parker, Lisan [St. Jude Children' s Research Hospital; Guo, Yun-Kai [St. Jude Children' s Research Hospital; Miller, Darla R [ORNL; Swanson, Douglas J [ORNL; Yoo, Tai-June [Neuroscience Institute, Memphis, TN; Goldowitz, Daniel [University of Tennessee Health Science Center, Memphis; Zuo, Jian [St. Jude Children' s Research Hospital

    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.

  19. Untargeted viral mutagenesis is not found in X-irradiated monkey cells

    International Nuclear Information System (INIS)

    Lytle, C.D.; Carney, P.G.; Lee, W.; Bushar, H.F.

    1988-01-01

    The existence of untargeted viral mutagenesis in X-irradiated cells was investigated in a mammalian virus/cell system, where a low level of such viral mutagenesis can be demonstrated in UV-irradiated cells. In the positive control experiment UV-elicited mutagenesis was shown with cell exposures of 5, 10 and 15 J/m 2 and a delay of 24 h between cell irradiation and infection with unirradiated herpes simplex virus. Although X-ray doses of 1, 3 and 10 Gy elicit enhanced reactivation of UV-irradiated virus, no untargeted mutagenesis for any X-ray dose at post-irradiation infection times of 0, 24 or 72 h was observed in this study. Thus untargeted mutagenesis of herpes simplex virus was not demonstrated in X-irradiated monkey cells, under conditions where X-ray-enhanced reactivation occurs and where untargeted mutagenesis in UV-irradiated cells occurs. (author)

  20. Stationary-state mutagenesis in Escherichia coli: a model

    Indian Academy of Sciences (India)

    conjugational recombination, where the RecBCD enzyme is generally believed to be loaded on to DNA from a double- stranded end and uses its nuclease and helicase activities to generate an invasive 3H single-strand end. However, the effect of mutations in the strand transfer genes (ruvA, ruvB,. ruvC and recG) on SSM ...

  1. Genotoxin Induced Mutagenesis in the Model Plant Physcomitrella patens

    Czech Academy of Sciences Publication Activity Database

    Holá, Marcela; Kozák, Jaroslav; Vágnerová, Radka; Angelis, Karel

    -, ID 535049 (2013) ISSN 2314-6133 R&D Projects: GA ČR GA13-06595S; GA MŠk(CZ) LD13006 Institutional support: RVO:61389030 ; RVO:61388963 Keywords : DNA-DAMAGE * COMET ASSAY * ARABIDOPSIS-THALIANA Subject RIV: EB - Genetics ; Molecular Biology

  2. Stationary-state mutagenesis in Escherichia coli: a model

    Indian Academy of Sciences (India)

    nature is essential for finally settling the controversy between Darwinism and the Lamarckian possibilities of generating exclusively growth-promoting mutations in nondividing cells. These mechanisms seem to provide an important tool of evolvability to organisms exposed to variable environment and appear to contribute ...

  3. In vitro mutagenesis of commercial fern, Asplenium nidus from spores

    International Nuclear Information System (INIS)

    Norazlina Noordin

    2004-01-01

    Asplenium is a largest, most diverse fern genera. One of the common species is Asplenium nidus, well known as Bird's-nest fern, a medium to large fern with erect, stout, unbranched rhizomes. In creating variability of ferns for the benefit of the ornamental plant industry, in vitro mutagenesis is used. In this study, spores of Asplenium nidus were collected from frond bearing mature sporangia. Spores were cultured in modified 1/2 MS basal medium supplemented with various combinations of 6-Benzylaminopurine (BAP) and Naphtalene Acetic Acid (NAA). Spore cultures were incubated in incubation room at 24 degree C with 16 hours photoperiod (3500 lux). It was found that, the most effective combinations were 1 mg/1 BAP + 0. 1 mg/1 NAA and 2mg/1 BAP + 0. 1 mg/1 NAA. Prothallus was formed after 10 days of cultures and gametophytes were formed 1 month later. These gametophytes were irradiated with Gamma ray at doses of 0, 20, 90, 120, 150 and 180 Gy. From the preliminary result obtained from this study, for generating variations and desired phenotypic expression for Asplenium nidus, recommended doses for in vitro mutagenesis using spores are between 90 Gy to 150 Gy. Gametophytes were subcultured at monthly interval to ensure further development and propagation. Frequent monitoring for any changes in the morphology of the irradiated Asplenium nidus plants were carried out. (Author)

  4. Mechanisms of Base Substitution Mutagenesis in Cancer Genomes

    Directory of Open Access Journals (Sweden)

    Albino Bacolla

    2014-03-01

    Full Text Available 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.

  5. Mechanisms of base substitution mutagenesis in cancer genomes.

    Science.gov (United States)

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

    2014-03-05

    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.

  6. Crowding depression of UV-mutagenesis in E. coli

    International Nuclear Information System (INIS)

    Bockrath, R.; Harper, D.; Kristoff, S.; Stanford Univ., CA

    1980-01-01

    Strains of E. coli Br were exposed to UV radiation and assayed for reversion mutation, using a standard selection medium. If more irradiated bacteria were assayed per petri dish, a proportional increase in the number of indicated reversion mutants was oud only up to a limiting plating density. Beyond a density of about 10 8 viable bacteria per petri dish, the number of indicated revertants per viable bacteriy assayed (the mutation frequency) decreased as the plating density was increased. The crowding depression of mutagenesis was more severe for de novo and converted suppressor mutations, the mutation frequency being reduced 100-fold at a plating density of about 6 x 10 9 viable bacteria per plate. The effect on backmutation was 10 times less. Crowding depression of mutagenesis occured in excision-proficient and -deficient strains, with identical effects in the 2 strains on de novo and converted suppressor mutation, but different effects on backmutations. There were no accompanying effects on viability. Irreversible loss of potential mutants during crowded growth was indicated in wash-off experiments. The kinetics suggested a half-life of approximately 1 h. Kinetics for accumulation by the bacteria of the limiting metabolite (tyrosine) on the assay plate indicated a short period of time for protein synthesis, but direct examination of the proteins synthesized during early growth on a crowded plate demonstrated successful induction of recA protein. (orig.)

  7. Tradescantia bioassays as monitoring systems for environmental mutagenesis: a review

    International Nuclear Information System (INIS)

    Rodrigues, G.S.; Ma, T.H.; Pimentel, D.; Weinstein, L.H.

    1997-01-01

    Since the early studies on the genetic effects of chemical and physical agents, species and clones of Tradescantia have been used as experimental subjects, by virtue of a series of favorable genetic characteristics. Bearing just six pairs (2n = 12) of large, easily observable chromosomes, cells from almost every part of the plant, from the root tips to the developing pollen tube, yield excellent material for cytogenetic studies. As a consequence of the intensive use of Tradescantia in genetic studies, a series of genetic characteristics have been found that offer opportunities for the detection of agents affecting the stability of the genome. At least five such characteristics have been selected as endpoints for the establishment of assays to evaluate mutagenesis. Three of these, root-tip mitosis, pollen-tube, and microspore mitosis are essentially chromosome aberration assays, wherein one observes and evaluates the visible damage in the chromosomes. A fourth, the stamen-hair mutation assay (Trad-SHM), is a point mutation mitotic assay based on the expression of a recessive gene for flower color in heterozygous plants. The fifth assay is a cytogenetic test based on the formation of micronuclei (Trad-MCN) that result from chromosome breakage in the meiotic pollen mother cells. This article examines the characteristics and fundamentals of the Trad-MCN and the Trad-SHM assays and reviews the results obtained to date with these systems in the assessment of environmental mutagenesis. (author)

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

    Science.gov (United States)

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

    2011-01-01

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

  9. Tissue culture and mutagenesis of rain lily (zephyranthes)

    International Nuclear Information System (INIS)

    Mohd Nazir Basiran; Zaiton Ahmad; Shakinah Salleh; Shuhaimi Shamsudin; Aiza Shaliha Jamaludin

    2004-01-01

    There are three varieties of Zephyranthes used widely in landscaping due to their robust growth and attractive flowers in pink, yellow and white. Both in vivo and in vitro mutagenesis are an effective approach to increase the flower colour variations of Zephyranthes. In vitro propagation for the three varieties was attempted by using the induction medium developed by Sachar and Kapoor in 1959. The medium contains I ma of each indole 3-acetic acid (IAA), indole 3-butyric acid (IBA) and kinetin. Following surface sterilization of bulb scales, 17.8%, 10.5% and 10.7% of pink, white and yellow varieties respectively, were able to form small bulblets on the induction media. Further development of these bulblets into plantlets was also achieved on the same medium. Work is now being carried out to improve the efficiency of bulblet regeneration. Mutagenesis of Zephyranthes was initiated from bulbs of the pink varieties to develop new varieties with attractive combinations of flower colour and forms, shelf life and growth habits. These bulbs were irradiated using a gamma cell with a 60 Co source. Three variants with different flower colour and morphology have been achieved so far and are now being propagated in the nursery. (Author)

  10. Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis.

    Science.gov (United States)

    Tagami, Uno; Shimba, Nobuhisa; Nakamura, Mina; Yokoyama, Kei-Ichi; Suzuki, Ei-Ichiro; Hirokawa, Takatsugu

    2009-12-01

    Transglutaminases (TGases) are used in fields such as food and pharmaceuticals. Unlike other TGases, microbial transglutaminase (MTG) activity is Ca(2+)-independent, broadening its application. Here, a three-dimensional docking model of MTG binding to a peptide substrate, CBZ-Gln-Gly, was simulated. The data reveal CBZ-Gln-Gly to be stretched along the MTG active site cleft with hydrophobic and/or aromatic residues interacting directly with the substrate. Moreover, an oxyanion binding site for TGase activity may be constructed from the amide groups of Cys64 and/or Val65. Alanine mutagenesis verified the simulated binding region and indicated that large molecules can be widely recognized on the MTG cleft.

  11. Improving isopropanol tolerance and production of Clostridium beijerinckii DSM 6423 by random mutagenesis and genome shuffling

    NARCIS (Netherlands)

    Máté De Gérando, H.; Fayolle-Guichard, F.; Rudant, L.; Millah, S.K.; Monot, F.; Ferreira, Nicolas Lopes; López-Contreras, A.M.

    2016-01-01

    Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of

  12. Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Wood, R.D.; Hutchinson, F. (Yale Univ., New Haven, CT (USA). Dept. of Molecular Biophysics and Biochemistry)

    1984-03-05

    Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr/sup +/ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one or two mutant phage per mutant burst. From the results of a series of experiments with various mutant host cells, a major pathway of non-targeted mutagenesis by ultraviolet light was proposed which acts in addition to ''SOS induction''. This pathway involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.

  13. Modeling and Re-Engineering of Azotobacter vinelandii Alginate Lyase to Enhance Its Catalytic Efficiency for Accelerating Biofilm Degradation.

    Directory of Open Access Journals (Sweden)

    Chul Ho Jang

    Full Text Available Alginate is known to prevent elimination of Pseudomonas aeruginosa biofilms. Alginate lyase (AlgL might therefore facilitate treatment of Pseudomonas aeruginosa-infected cystic fibrosis patients. However, the catalytic activity of wild-type AlgL is not sufficiently high. Therefore, molecular modeling and site-directed mutagenesis of AlgL might assist in enzyme engineering for therapeutic development. AlgL, isolated from Azotobacter vinelandii, catalyzes depolymerization of alginate via a β-elimination reaction. AlgL was modeled based on the crystal structure template of Sphingomonas AlgL species A1-III. Based on this computational analysis, AlgL was subjected to site-directed mutagenesis to improve its catalytic activity. The kcat/Km of the K194E mutant showed a nearly 5-fold increase against the acetylated alginate substrate, as compared to the wild-type. Double and triple mutants (K194E/K245D, K245D/K319A, K194E/K245D/E312D, and K194E/K245D/K319A were also prepared. The most potent mutant was observed to be K194E/K245D/K319A, which has a 10-fold improved kcat value (against acetylated alginate compared to the wild-type enzyme. The antibiofilm effect of both AlgL forms was identified in combination with piperacillin/tazobactam (PT and the disruption effect was significantly higher in mutant AlgL combined with PT than wild-type AlgL. However, for both the wild-type and K194E/K245D/K319A mutant, the use of the AlgL enzyme alone did not show significant antibiofilm effect.

  14. Somatic mutagenesis with a Sleeping Beauty transposon system leads to solid tumor formation in zebrafish.

    Directory of Open Access Journals (Sweden)

    Maura McGrail

    2011-04-01

    Full Text Available Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700-6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.

  15. Creating Sunflower Mutant Lines (Helianthus Annuus L.) Using Induced Mutagenesis

    International Nuclear Information System (INIS)

    Encheva, J.

    2009-01-01

    Immature sunflower zygotic embryos of sunflower fertility restorer line 374 R were treated with ultrasound and gamma radiation before plating embryos to culture medium. All plants were isolated and self-pollinated for several generations. New sunflower forms with inherited morphological and biochemical changes were obtained. The genetic changes occurring during the mutation procedure included fourteen morphological and biochemical characters. In comparison to the check line 374 R, decreasing of the mean value of the indexes was registered for 33 % of the total number of characters and vise verse, significant increasing was observed for 60 %. Mutation for resistance to the local population of Orobanche cumana race A-E was obtained from the susceptible Bulgarian control line 374 R. Two investigated mutant lines possessed 100 % resistance to Orobanche and stable inheritance in the next generations. Our results showed that induced mutagenesis in sunflower can be successfully used to develop new lines useful for heterosis breeding

  16. Radiation induced DNA damage and repair in mutagenesis

    International Nuclear Information System (INIS)

    Strniste, G.F.; Chen, D.J.; Okinaka, R.T.

    1987-01-01

    The central theme in cellular radiobiological research has been the mechanisms of radiation action and the physiological response of cells to this action. Considerable effort has been directed toward the characterization of radiation-induced DNA damage and the correlation of this damage to cellular genetic change that is expressed as mutation or initiating events leading to cellular transformation and ultimately carcinogenesis. In addition, there has been a significant advancement in their understanding of the role of DNA repair in the process of mutation leading to genetic change in cells. There is extensive literature concerning studies that address radiation action in both procaryotic and eucaryotic systems. This brief report will make no attempt to summarize this voluminous data but will focus on recent results from their laboratory of experiments in which they have examined, at both the cellular and molecular levels, the process of ionizing radiation-induced mutagenesis in cultured human cells

  17. Scientific projection paper for mutagenesis, transformation and cell killing

    International Nuclear Information System (INIS)

    Todd, P.

    1980-01-01

    Our knowledge about mutagenesis, transformation, and cell killing by ionizing radiation consists of large bodies of data, which are potentially useful in terms of application to human risk assessment and to the constructive use of radiation, as in cancer treatment. The three end-points discussed above are united by at least five significant concepts in radiation research strategy: (1) The inter-relationships among the important end-points, mutation, carcinogenesis, and cell killing. Research on one is meaningful only in the context of information about the other two. (2) The interaction of radiations with other agents in producing these end-points. (3) The mechanisms of action of other environmental mutagenic, carcinogenic, and cytotoxic agents. (4) The use of repair deficient human mutant cells. (5) The study of radiation damage mechanisms. There is no better way to extrapolate laboratory data to the clinical and public worlds than to understand the underlying biological mechanisms that produced the data

  18. Results and perspectives of mutagenesis applied to durum wheat

    International Nuclear Information System (INIS)

    Bagnara, D.

    1975-01-01

    A review is made of the main aspects and problems of mutagenesis applied to the breeding of durum wheat (Triticum turgidum ssp. durum). Features and type of action of the main physical and chemical mutagens are considered: a comparison is also made between the two classes of mutagens, on the basis of results so far achieved. Mentions is then made of methods of treatment; parts of plant which can be treated; growing of treated material in segregating generations: data to be successively recorded. Methods of estimating mutation frequency and the problem of arising chimerical tissues and its possible overcoming are also discussed. Examination is made of some special effects of mutagens, namely: induction of translocations; diploidization of polyploids; induction of haploids and aneuploids; genetic analysis of specific loci; induction of male sterility. Finally, results are reviewed concerning induction and utilization, either as varieties or in cross breeding programmes, of mutants for characters of agronomic interest. (Bagnara, D.)

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

    Directory of Open Access Journals (Sweden)

    Alexandro Rodríguez-Rojas

    2015-10-01

    Full Text Available 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.

  20. Mapping of epitopes for autoantibodies to the Type 1 diabetes autoantigen IA-2 by peptide phage display and molecular modelling: Overlap of antibody and T-cell determinants

    DEFF Research Database (Denmark)

    A. Dromey, James; Weenink, Sarah M.; Peters, Günther H.J.

    2004-01-01

    IA-2 is a major target of autoimmunity in type 1 diabetes. IA-2 responsive T cells recognize determinants within regions represented by amino acids 787–817 and 841–869 of the molecule. Epitopes for IA-2 autoantibodies are largely conformational and not well defined. In this study, we used peptide......, and aromatic residues and amino acids contributing to the epitope investigated using site-directed mutagenesis. Mutation of each of amino acids Asn858, Glu836, and Trp799 reduced 96/3 Ab binding by >45%. Mutations of these residues also inhibited binding of serum autoantibodies from IA-2 Ab-positive type 1...... phage display and homology modeling to characterize the epitope of a monoclonal IA-2 Ab (96/3) from a human type 1 diabetic patient. This Ab competes for IA-2 binding with Abs from the majority of patients with type 1 diabetes and therefore binds a region close to common autoantibody epitopes. Alignment...

  1. Catalytic soman scavenging by Y337A/F338A acetylcholinesterase mutant assisted with novel site-directed aldoximes

    Science.gov (United States)

    Kovarik, Zrinka; Hrvat, Nikolina Maček; Katalinić, Maja; Sit, Rakesh K.; Paradyse, Alexander; Žunec, Suzana; Musilek, Kamil; Fokin, Valery V.; Taylor, Palmer; Radić, Zoran

    2016-01-01

    Exposure to the nerve agent soman is difficult to treat due to the rapid dealkylation of soman-acetylcholinesterase (AChE) conjugate known as aging. Oxime antidotes commonly used to reactivate organophosphate inhibited AChE are ineffective against soman, while the efficacy of the recommended nerve agent bioscavenger butyrylcholinesterase is limited by strictly stoichiometric scavenging. To overcome this limitation, we tested ex vivo, in human blood, and in vivo, in soman exposed mice, the capacity of aging-resistant human AChE mutant Y337A/F338A in combination with oxime HI-6 to act as a catalytic bioscavenger of soman. HI-6 was previously shown in vitro to efficiently reactivate this mutant upon soman, as well as VX, cyclosarin, sarin and paraoxon inhibition. We here demonstrate that ex vivo, in whole human blood, 1 μM soman was detoxified within 30 minutes when supplemented with 0.5 μM Y337A/F338A AChE and 100 μM HI-6. This combination was further tested in vivo. Catalytic scavenging of soman in mice improved the therapeutic outcome and resulted in the delayed onset of toxicity symptoms. Furthermore, in a preliminary in vitro screen we identified an even more efficacious oxime than HI-6, in a series of forty-two pyridinium aldoximes, and five imidazole 2-aldoxime N-propyl pyridinium derivatives. One of the later imidazole aldoximes, RS-170B, was a 2–3 –fold more effective reactivator of Y337A/F338A AChE than HI-6 due to the smaller imidazole ring, as indicated by computational molecular models, that affords a more productive angle of nucleophilic attack. PMID:25835984

  2. Topology of AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, determined by site-directed fluorescence labeling.

    Science.gov (United States)

    Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C; Abe, Keietsu

    2007-10-01

    The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of L-aspartate (Asp) with release of L-alanine (Ala) and CO(2). The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an L-aspartate-beta-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.

  3. Model photo reaction centers via genetic engineering

    Energy Technology Data Exchange (ETDEWEB)

    Zhiyu Wang; DiMagno, T.J.; Popov, M.; Norris, J.R. [Argonne National Lab., IL (United States)]|[Chicago Univ., IL (United States). Dept. of Chemistry; Chikin Chan; Fleming, G. [Chicago Univ., IL (United States). Dept. of Chemistry; Jau Tang; Hanson, D.; Schiffer, M. [Argonne National Lab., IL (United States)

    1992-12-31

    A series of reaction centers of Rhodococcus capsulatus isolated from a set of mutated organisms modified by site-directed mutagenesis at residues M208 and L181 are described. Changes in the amino acid at these sites affect both the energetics of the systems as well as the chemical kinetics for the initial ET event. Two empirical relations among the different mutants for the reduction potential and the ET rate are presented.

  4. Model photo reaction centers via genetic engineering

    Energy Technology Data Exchange (ETDEWEB)

    Zhiyu Wang; DiMagno, T.J.; Popov, M.; Norris, J.R. (Argonne National Lab., IL (United States) Chicago Univ., IL (United States). Dept. of Chemistry); Chikin Chan; Fleming, G. (Chicago Univ., IL (United States). Dept. of Chemistry); Jau Tang; Hanson, D.; Schiffer, M. (Argonne National Lab., IL (United States))

    1992-01-01

    A series of reaction centers of Rhodococcus capsulatus isolated from a set of mutated organisms modified by site-directed mutagenesis at residues M208 and L181 are described. Changes in the amino acid at these sites affect both the energetics of the systems as well as the chemical kinetics for the initial ET event. Two empirical relations among the different mutants for the reduction potential and the ET rate are presented.

  5. The crystal structure, mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad.

    Science.gov (United States)

    Rydel, Timothy J; Williams, Jennifer M; Krieger, Elysia; Moshiri, Farhad; Stallings, William C; Brown, Sherri M; Pershing, Jay C; Purcell, John P; Alibhai, Murtaza F

    2003-06-10

    Patatin is a nonspecific lipid acyl hydrolase that accounts for approximately 40% of the total soluble protein in mature potato tubers, and it has potent insecticidal activity against the corn rootworm. We determined the X-ray crystal structure of a His-tagged variant of an isozyme of patatin, Pat17, to 2.2 A resolution, employing SeMet multiwavelength anomalous dispersion (MAD) phasing methods. The patatin crystal structure has three molecules in the asymmetric unit, an R-factor of 22.0%, and an R(free) of 27.2% (for 10% of the data not included in the refinement) and includes 498 water molecules. The structure notably revealed that patatin has a Ser-Asp catalytic dyad and an active site like that of human cytosolic phospholipase A(2) (cPLA(2)) [Dessen, A., et al. (1999) Cell 97, 349-360]. In addition, patatin has a folding topology related to that of the catalytic domain of cPLA(2) and unlike the canonical alpha/beta-hydrolase fold. The structure confirms our site-directed mutagenesis and bioactivity data that initially suggested patatin possessed a Ser-Asp catalytic dyad. Alanine-scanning mutagenesis revealed that Ser77 and Asp215 were critical for both esterase and bioactivity, consistent with prior work implicating a Ser residue [Strickland, J. H., et al. (1995) Plant Physiol. 109, 667-674] and a Ser-Asp dyad [Hirschberg, H. J. H. B., et al. (2001) Eur. J. Biochem. 268, 5037-5044] in patatin's catalytic activity. The crystal structure aids the understanding of other structure-function relationships in patatin. Patatin does not display interfacial activation, a hallmark feature of lipases, and this is likely due to the fact that it lacks a flexible lid that can shield the active site.

  6. Characterizing the Hot Spots Involved in RON-MSPβ Complex Formation Using In Silico Alanine Scanning Mutagenesis and Molecular Dynamics Simulation

    Directory of Open Access Journals (Sweden)

    Omid Zarei

    2017-04-01

    Full Text Available Purpose: Implication of protein-protein interactions (PPIs in development of many diseases such as cancer makes them attractive for therapeutic intervention and rational drug design. RON (Recepteur d’Origine Nantais tyrosine kinase receptor has gained considerable attention as promising target in cancer therapy. The activation of RON via its ligand, macrophage stimulation protein (MSP is the most common mechanism of activation for this receptor. The aim of the current study was to perform in silico alanine scanning mutagenesis and to calculate binding energy for prediction of hot spots in protein-protein interface between RON and MSPβ chain (MSPβ. Methods: In this work the residues at the interface of RON-MSPβ complex were mutated to alanine and then molecular dynamics simulation was used to calculate binding free energy. Results: The results revealed that Gln193, Arg220, Glu287, Pro288, Glu289, and His424 residues from RON and Arg521, His528, Ser565, Glu658, and Arg683 from MSPβ may play important roles in protein-protein interaction between RON and MSP. Conclusion: Identification of these RON hot spots is important in designing anti-RON drugs when the aim is to disrupt RON-MSP interaction. In the same way, the acquired information regarding the critical amino acids of MSPβ can be used in the process of rational drug design for developing MSP antagonizing agents, the development of novel MSP mimicking peptides where inhibition of RON activation is required, and the design of experimental site directed mutagenesis studies.

  7. Mutagenesis Objective Search and Selection Tool (MOSST: an algorithm to predict structure-function related mutations in proteins

    Directory of Open Access Journals (Sweden)

    Asenjo Juan A

    2011-04-01

    Full Text Available Abstract Background Functionally relevant artificial or natural mutations are difficult to assess or predict if no structure-function information is available for a protein. This is especially important to correctly identify functionally significant non-synonymous single nucleotide polymorphisms (nsSNPs or to design a site-directed mutagenesis strategy for a target protein. A new and powerful methodology is proposed to guide these two decision strategies, based only on conservation rules of physicochemical properties of amino acids extracted from a multiple alignment of a protein family where the target protein belongs, with no need of explicit structure-function relationships. Results A statistical analysis is performed over each amino acid position in the multiple protein alignment, based on different amino acid physical or chemical characteristics, including hydrophobicity, side-chain volume, charge and protein conformational parameters. The variances of each of these properties at each position are combined to obtain a global statistical indicator of the conservation degree of each property. Different types of physicochemical conservation are defined to characterize relevant and irrelevant positions. The differences between statistical variances are taken together as the basis of hypothesis tests at each position to search for functionally significant mutable sites and to identify specific mutagenesis targets. The outcome is used to statistically predict physicochemical consensus sequences based on different properties and to calculate the amino acid propensities at each position in a given protein. Hence, amino acid positions are identified that are putatively responsible for function, specificity, stability or binding interactions in a family of proteins. Once these key functional positions are identified, position-specific statistical distributions are applied to divide the 20 common protein amino acids in each position of the protein

  8. Role of the RecF gene product in UV mutagenesis of lambda phage

    International Nuclear Information System (INIS)

    Wood, R.D.; Stein, J.

    1986-01-01

    E. coli recF mutants have a greatly reduced capacity for Weigle mutagenesis of ultraviolet light-irradiated lambda phage. A recF 332::Tn3 mutation was introduced into an E. coli recA441 lex A51 strain which constitutively expresses SOS functions. Weigle mutagenesis of phage lambda could occur in the resulting strain in the absence of host cell irradiation, and was increased when the recA441 (tif) allele was activated of recF strains to support Weigle mutagenesis can therefore be ascribed to a defect in expression of SOS functions after irradiation. (orig.)

  9. X-ray structure at 1.75 resolution of a norovirus 3C protease linked to an active site-directed peptide inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, Jon [University of Southampton, England; Coates, Leighton [ORNL; Hussey, Robert [University of Southampton, England

    2010-01-01

    Noroviruses are recognized universally as the most important cause of human epidemic non-bacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.75 resolution, following initial MAD phasing with a selenomethionine derivative. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, based on a 3C protease cleavage recognition sequences in the 200kDa polyprotein substrate, reacts covalently through its propenylethylester group (X) with the active site nucleophile, Cys 139. The 3C protease-inhibitor structure permits, for the first time, the identification of substrate recognition and binding groups and provides important new information for the development of antiviral prophylactics.

  10. Reverse genetics through random mutagenesis in Histoplasma capsulatum

    Directory of Open Access Journals (Sweden)

    Rappleye Chad A

    2009-11-01

    Full Text Available Abstract Background The dimorphic fungal pathogen Histoplasma capsulatum causes respiratory and systemic disease in humans and other mammals. Progress in understanding the mechanisms underlying the biology and the pathogenesis of Histoplasma has been hindered by a shortage of methodologies for mutating a gene of interest. Results We describe a reverse genetics process that combines the random mutagenesis of Agrobacterium-mediated transformation with screening techniques to identify targeted gene disruptions in a collection of insertion mutants. Isolation of the desired mutant is accomplished by arraying individual clones from a pool and employing a PCR-addressing method. Application of this procedure facilitated the isolation of a cbp1 mutant in a North American type 2 strain, a Histoplasma strain recalcitrant to gene knock-outs through homologous recombination. Optimization of cryopreservation conditions allows pools of mutants to be banked for later analysis and recovery of targeted mutants. Conclusion This methodology improves our ability to isolate mutants in targeted genes, thereby facilitating the molecular genetic analysis of Histoplasma biology. The procedures described are widely applicable to many fungal systems and will be of particular interest to those for which homologous recombination techniques are inefficient or do not currently exist.

  11. A wheat cold resistance mutant derived from space mutagenesis

    International Nuclear Information System (INIS)

    Li Peng; Sun Mingzhu; Zhang Fengyun; Gao Guoqiang; Qiu Denglin; Li Xinhua

    2012-01-01

    A cold resistance mutant, obtained by spaceflight mutagenesis on the seeds of wheat variety Han6172, and the DNA of cold resistance mutant and contrast Han6172 were compared by SRAP technique. 380 pairs of primers were screened, 6 pairs of them had polymorphisms between mutant and contrast, the rate was 1.58%, and this data indicated that there are no obvious DNA differences between mutant and contrast Six specific fragments were obtained, 3 fragments of them were amplified in mutant. Homology analysis in GenBank showed that Me3-Em7-Mt, Me4-Em11-CK, Me7-Em19-CK and Me6-Em9-Mt all had homologous sequences with wheat chromosome 3B-specific BAC library, and this result indicated that the gene and regulator sequences associated with mutant cold resistance might locate on 3B chromosome. It was speculated that space mutation induced the mutation of 3B chromosome primary structure, and influenced the expressions of cold resistance genes, which resulted in the mutation of cold resistance ability. (authors)

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

    Science.gov (United States)

    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.

  13. Retroviral Vectors for Analysis of Viral Mutagenesis and Recombination

    Directory of Open Access Journals (Sweden)

    Jonathan M.O. Rawson

    2014-09-01

    Full Text Available 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.

  14. ENU Mutagenesis in Mice Identifies Candidate Genes For Hypogonadism

    Science.gov (United States)

    Weiss, Jeffrey; Hurley, Lisa A.; Harris, Rebecca M.; Finlayson, Courtney; Tong, Minghan; Fisher, Lisa A.; Moran, Jennifer L.; Beier, David R.; Mason, Christopher; Jameson, J. Larry

    2012-01-01

    Genome-wide mutagenesis was performed in mice to identify candidate genes for male infertility, for which the predominant causes remain idiopathic. Mice were mutagenized using N-ethyl-N-nitrosourea (ENU), bred, and screened for phenotypes associated with the male urogenital system. Fifteen heritable lines were isolated and chromosomal loci were assigned using low density genome-wide SNP arrays. Ten of the fifteen lines were pursued further using higher resolution SNP analysis to narrow the candidate gene regions. Exon sequencing of candidate genes identified mutations in mice with cystic kidneys (Bicc1), cryptorchidism (Rxfp2), restricted germ cell deficiency (Plk4), and severe germ cell deficiency (Prdm9). In two other lines with severe hypogonadism candidate sequencing failed to identify mutations, suggesting defects in genes with previously undocumented roles in gonadal function. These genomic intervals were sequenced in their entirety and a candidate mutation was identified in SnrpE in one of the two lines. The line harboring the SnrpE variant retains substantial spermatogenesis despite small testis size, an unusual phenotype. In addition to the reproductive defects, heritable phenotypes were observed in mice with ataxia (Myo5a), tremors (Pmp22), growth retardation (unknown gene), and hydrocephalus (unknown gene). These results demonstrate that the ENU screen is an effective tool for identifying potential causes of male infertility. PMID:22258617

  15. Targeted mutagenesis using CRISPR/Cas system in medaka

    Directory of Open Access Journals (Sweden)

    Satoshi Ansai

    2014-04-01

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

  16. Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Rongming; Liang, Liya; Garst, Andrew D.; Choudhury, Alaksh; Nogué, Violeta Sànchez i.; Beckham, Gregg T.; Gill, Ryan T.

    2018-05-01

    Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)H metabolism, and the aldehyde reduction system. Specific mutants that conferred increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. We tested the strain with positive combinatorial mutations for 3-hydroxypropionic acid (3HP) production under high furfural and high acetate hydrolysate fermentation, which demonstrated a 7- and 8-fold increase in 3HP productivity relative to the parent strain, respectively.

  17. Oligonucleotide-directed mutagenesis for precision gene editing.

    Science.gov (United States)

    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. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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

    Science.gov (United States)

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

    2015-10-01

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

  19. In vitro mutagenesis for the improvement of Josapine pineapple

    International Nuclear Information System (INIS)

    Rusli Ibrahim; Amir Hamzah

    2006-01-01

    Pineapple is the most important fruit in terms of revenue earner in Malaysia. There are about 10,000 ha cultivated with this fruit and half of this is owned by estates and planted for the canning industry. The export of canned pineapple is about 2 million standard cases annually valued at RM 60 million, while the export of fresh pineapple is about 40,000 tonnes worth about RM 10 million. The industry for canning is however, an ailing industry with production on the decline since the 70s. Somaclonal variations and induced mutation using irradiation in breeding are least invasive in changes to genetic make-up of an established variety and will be useful for improving the pineapple varieties. The use of tissue culture to generate somaclones with minute genetic changes that do not damage the overall varietal identity would be the most suitable tool to improve the variety. Protocols for the production of tissue culture plantlets of pineapple using bioreactor technology has been developed and proved to be much more efficient and productive compared to conventional method. In vitro mutagenesis using adventitious buds had produced new plants with smooth leaves, vigorous growth and ornamental-like characters. A total of 30,000 plants derived from tissue culture will be planted and screened in the field for the improvement of Josapine pineapple against bacterial heart rot disease and multiple crown. (Author)

  20. Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK.

    Science.gov (United States)

    Berezuk, Alison M; Goodyear, Mara; Khursigara, Cezar M

    2014-08-22

    In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, and the C terminus (FtsKC) is a well characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis

    Science.gov (United States)

    Xu, Zhong; Wang, Yemin; Chater, Keith F.; Ou, Hong-Yu; Xu, H. Howard; Deng, Zixin

    2017-01-01

    ABSTRACT Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5 transposition system and employed it to mutagenize the model species Streptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5 transposition targeting in the high-GC Streptomyces genome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement. IMPORTANCE High-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5 for the study of secondary metabolism and its

  2. Mutagenesis of bacteriophage T7 and T7 DNA by alkylation damage.

    OpenAIRE

    Masker, W E; Dodson, L A; Maupin, M

    1985-01-01

    We have developed a new assay for in vitro mutagenesis of bacteriophage T7 DNA that measures the generation of mutations in the specific T7 gene that codes for the phage ligase. This assay was used to examine mutagenesis caused by in vitro DNA synthesis in the presence of O6-methylguanosine triphosphate. Reversion of one of the newly generated ligase mutants by ethyl methanesulfonate was also tested.

  3. Mutagenesis of bacteriophage T7 and T7 DNA by alkylation damage.

    Science.gov (United States)

    Masker, W E; Dodson, L A; Maupin, M

    1985-01-01

    We have developed a new assay for in vitro mutagenesis of bacteriophage T7 DNA that measures the generation of mutations in the specific T7 gene that codes for the phage ligase. This assay was used to examine mutagenesis caused by in vitro DNA synthesis in the presence of O6-methylguanosine triphosphate. Reversion of one of the newly generated ligase mutants by ethyl methanesulfonate was also tested. PMID:3903213

  4. Mutagenesis and haploid culture for disease resistance in Brassica napus

    International Nuclear Information System (INIS)

    MacDonald, M.V.; Ahmad, I.; Ingram, D.S.

    1990-01-01

    Full text: Most winter oilseed rape cultivars share parentage and therefore show little genetic diversity. There is no known resistance to Alternaria spp. in oilseed rape or in any related Brassica species. Experiments with tissue culture yielded only transient, non-genetic resistance. Therefore, mutagenesis may be used to generate heritable resistance to Alternaria spp. Gamma irradiation was applied to seeds of 'Bienvenue', secondary embryoids of cvs 'Primor' and 'Rapora', and buds of cvs 'Primor' and 'Ariana'. Isolated microspores from cv 'Ariana' and rapid cycling B. napus were also treated. The doses used ranged from 0-100 Gy for isolated microspores and buds, up to 600 Gy for seeds and 960 Gy for secondary embryoids. EMS was used to treat seeds of line WRG-42 (supplied by Nickersons RPB) and microspores of cv 'Bienvenue' and rapid cycling B. napus. Seeds were treated with up to 2.0% EMS for 0.2 h. before plating them on the culture medium. Seed irradiation up to 600 Gy did not reduce germination. M 1 and M 2 progenies were tested both in the laboratory and in field trials, and none of these were found to be resistant to Alternaria. However, considerable variation for other characters was observed. Haploid cultures from these plants were extremely difficult to regenerate, and for this reason no regenerant plants have been tested for resistance. For irradiated secondary embryoids the regeneration capacity decreased with increasing dose. Regenerated plants have been tested for resistance to Alternaria, but stable resistance was not observed. Haploid cultures were obtained from irradiated buds, using both anther and microspore culture. Low irradiation treatment was beneficial to developing embryoids. Some regenerants have been obtained from EMS treated microspores and seeds. Four plants have repeatedly given increased levels of resistance to A. brassicicola, and progenies are being tested to determine the genetic nature of the resistance. (author)

  5. Arginine kinase in Toxocara canis: Exon-intron organization, functional analysis of site-directed mutants and evaluation of putative enzyme inhibitors.

    Science.gov (United States)

    Wickramasinghe, Susiji; Yatawara, Lalani; Nagataki, Mitsuru; Agatsuma, Takeshi

    2016-10-01

    To determine exon/intron organization of the Toxocara canis (T. canis) AK (TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants. Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame (1200 bp) of TCAK (wild type) was cloned into the BamH1/SalI site of pMAL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pMAL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25 °C for the forward reaction (phosphagen synthesis). Arginine kinase in T. canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542 bp to 2 500 bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The K m value of the mutant (Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The K m value of the mutant (Serine to Glycine) increased to 0.19 mM. The K m value (0.19 mM) of the double mutant (Alanine-Serine to Serine-Glycine) was slightly greater than in the wild-type (0.12 mM). In addition, several other chemicals were tested; including plant extract Azadiracta indica (A. indica), an aminoglycoside antibiotic (aminosidine), a citrus flavonoid glycoside (rutin) and a commercially available catechin mixture against TCAK. Green and black tea (1:10 dilution) produced 15% and 25% inhibition of TCAK, respectively. The extract of A. indica produced 5% inhibition of TCAK. Moreover, green and black tea produced a non-competitive type of inhibition and A. indica produced a mixed-type of inhibition on TCAK. Arginine kinase in T. canis has a seven-exon/six-intron gene

  6. Assay of new systems in vivo mutagenesis for determining the effects of low doses of ionizing radiation

    International Nuclear Information System (INIS)

    Bauluz, C.; Sierra, I.; Martin, L.; Real, A.; Vidania, R. de

    1997-01-01

    Ionizing radiation reacts directly and indirectly with the genetic material in living cells and produces DNA damage. Processing of this damage by correcting enzymes may result in appearing of mutations which, in turn, may lead to carcinogenesis. We have focused on the determination of in vivo mutagenesis induced after exposure to X-rays, aiming at establishing methods to evaluate the effect of low doses of radiation. In vivo mutagenesis has been addressed in the Muta Mouse model that carries a lacZ marker gene and provides a relatively simple assay of appearance of mutations. Mutation frequencies were determined in the lacZ gene copies recovered from mice irradiated with 1Gy or 4Gy of X-rays, acute or fractionated. Liver, spleen and bone marrow DNA samples were isolated at different times after irradiation, ranging from 1 day to 2 months, and evolution of mutations was studied. Results showed different responses depending on the organ and especially on the time of analysis, suggesting that the mutagenic process in vivo is much more complex than previously deduced from in vitro experiments. Therefore, determination of the relationship between dose and mutagenic effect in vivo will require additional studies. (author)

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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. [From random mutagenesis to precise genome editing: the development and evolution of genome editing techniques in Drosophila].

    Science.gov (United States)

    Su, Fang; Huang, Zong-liang; Guo, Ya-wen; Jiao, Ren-jie; Zi, Li; Chen, Jian-ming; Liu, Ji-yong

    2016-01-01

    Drosophila melanogaster, an important model organism for studying life science, has contributed more to the research of genetics, developmental biology and biomedicine with the development of genome editing techniques. Drosophila genome-editing techniques have evolved from random mutagenesis to precise genome editing and from simple mutant construction to diverse genome editing methods since the 20th century. Chemical mutagenesis, using Ethyl methanesulfonate (EMS), is an important technique to study gene function in forward genetics, however, the precise knockout of Drosophila genes could not be achieved. The gene targeting technology, based on homologous recombination, has accomplished the precise editing of Drosophila genome for the first time, but with low efficiency. The CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein)-mediated precise genome editing is simple, fast and highly efficient compared with the gene targeting technology in Drosophila. In this review, we focus on Drosophila gene knockout, and summarize the evolution of genome editing techniques in Drosophila, emphasizing the development and applications of gene targeting, zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and CRISPR/Cas9 techniques.

  9. Human plasma lecithin:cholesterol acyltransferase. Preparation and use of immobilized p-aminophenylarsenoxide as a catalytic site-directed covalent ligand in enzyme purification.

    Science.gov (United States)

    Zhou, G Y; Jauhiainen, M; Stevenson, K; Dolphin, P J

    1991-07-17

    A method is described for the preparation of p-aminophenylarsenoxide-linked carboxymethyl (CM) Bio-Gel A and its use as a specific, catalytic site-directed affinity chromatography ligand in the final stages of the purification of human plasma lecithin:cholesterol acyltransferase (LCAT) (EC 2.3.1.43). Previous mechanistic studies by us demonstrated that phenylarsenoxide derivatives, which are highly specific for vicinal thiols, could inhibit LCAT via a covalent interaction with the sulphydryl groups of the two catalytic cysteine residues and that this inhibition could be rapidly and completely reversed upon addition of 2,3-dimercaptopropanesulphonic acid. The ligand was covalently linked to CM Bio-Gel A activated through an N-hydroxysuccinyl ester formed by N-hydroxysuccinimide and dicyclohexylcarbodiimide in dry dimethyl sulphoxide; 87% of the added p-aminophenylarsenoxide was coupled to the CM Bio-Gel A in 3 h at 25 degrees C giving 2.3 mg of p-aminophenylarsenoxide per ml of gel. Homogeneous LCAT free of apo A-I, apo E, apo D and albumin was obtained in an 11% yield and purified 15,013-fold overall. A 13-fold purification was obtained by chromatography upon p-aminophenylarsenoxide-CM Bio-Gel A. This method is a useful final step in LCAT purification and will prove valuable in the purification of other proteins and compounds containing vicinal thiols.

  10. Exploitation of the S-layer self-assembly system for site directed immobilization of enzymes demonstrated for an extremophilic laminarinase from Pyrococcus furiosus

    Science.gov (United States)

    Tschiggerl, Helga; Breitwieser, Andreas; de Roo, Guy; Verwoerd, Theo; Scḧaffer, Christina; Sleytr, Uwe B.

    2015-01-01

    A fusion protein based on the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and the enzyme laminarinase (LamA) from Pyrococcus furiosus was designed and overexpressed in Escherichia coli. Due to the construction principle, the S-layer fusion protein fully retained the self-assembly capability of the S-layer moiety, while the catalytic domain of LamA remained exposed at the outer surface of the formed protein lattice. The enzyme activity of the S-layer fusion protein monolayer obtained upon recrystallization on silicon wafers, glass slides and different types of polymer membranes was determined colorimetrically and related to the activity of sole LamA that has been immobilized with conventional techniques. LamA aligned within the S-layer fusion protein lattice in a periodic and orientated fashion catalyzed twice the glucose release from the laminarin polysaccharide substrate in comparison to the randomly immobilized enzyme. In combination with the good shelf-life and the high resistance towards temperature and diverse chemicals, these novel composites are regarded a promising approach for site-directed enzyme immobilization. PMID:18035441

  11. Hydrogen/deuterium exchange mass spectrometry and site-directed disulfide cross-linking suggest an important dynamic interface between the two lysostaphin domains.

    Science.gov (United States)

    Lu, Hai-Rong; Gu, Mei-Gang; Huang, Qiang; Huang, Jin-jiang; Lu, Wan-Ying; Lu, Hong; Huang, Qing-Shan

    2013-04-01

    Lysostaphin is a peptidoglycan hydrolase secreted by Staphylococcus simulans. It can specifically lyse Staphylococcus aureus and is being tested as a novel antibacterial agent. The protein contains an N-terminal catalytic domain and a C-terminal cell wall targeting domain. Although the two domains from homologous enzymes were structurally determined, the structural organization of lysostaphin domains remains unknown. We used hydrogen/deuterium exchange mass spectrometry (H/DX-MS) and site-directed disulfide cross-linking to probe the interface between the lysostaphin catalytic and targeting domains. H/DX-MS-mediated comparison of peptides from full-length lysostaphin and the separated domains identified four peptides of lower solvent accessibility in the full-length protein. Cross-linking analysis using cysteine pair substitutions within those peptides showed that two pairs of cysteines can form disulfide bonds, supporting the domain association role of the targeted peptides. The cross-linked mutant exhibited a binding capacity to S. aureus that was similar to that of the wild-type protein but reduced bacteriolytic activity probably because of restraint in conformation. The diminished activity was further reduced with increasing NaCl concentrations that can cause contractions of bacterial peptidoglycan. The lytic activity, however, could be fully recovered by reducing the disulfide bonds. These results suggest that lysostaphin may require dynamic association of the two domains for coordinating substrate binding and target cleavage on the elastic peptidoglycan. Our study will help develop site-specific PEGylated lysostaphin to treat systemic S. aureus infections.

  12. Structure of Bacillus subtilis γ-glutamyltranspeptidase in complex with acivicin: diversity of the binding mode of a classical and electrophilic active-site-directed glutamate analogue

    Energy Technology Data Exchange (ETDEWEB)

    Ida, Tomoyo [Osaka University, Toyonaka, Osaka 560-0043 (Japan); Suzuki, Hideyuki [Kyoto Institute of Technology, Goshokaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585 (Japan); Fukuyama, Keiichi [Osaka University, Toyonaka, Osaka 560-0043 (Japan); Hiratake, Jun [Kyoto University, Uji, Kyoto 611-0011 (Japan); Wada, Kei, E-mail: keiwada@med.miyazaki-u.ac.jp [University of Miyazaki, Miyazaki 889-1692 (Japan); Osaka University, Toyonaka, Osaka 560-0043 (Japan)

    2014-02-01

    The binding modes of acivicin, a classical and an electrophilic active-site-directed glutamate analogue, to bacterial γ-glutamyltranspeptidases were found to be diverse. γ-Glutamyltranspeptidase (GGT) is an enzyme that plays a central role in glutathione metabolism, and acivicin is a classical inhibitor of GGT. Here, the structure of acivicin bound to Bacillus subtilis GGT determined by X-ray crystallography to 1.8 Å resolution is presented, in which it binds to the active site in a similar manner to that in Helicobacter pylori GGT, but in a different binding mode to that in Escherichia coli GGT. In B. subtilis GGT, acivicin is bound covalently through its C3 atom with sp{sup 2} hybridization to Thr403 O{sup γ}, the catalytic nucleophile of the enzyme. The results show that acivicin-binding sites are common, but the binding manners and orientations of its five-membered dihydroisoxazole ring are diverse in the binding pockets of GGTs.

  13. Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI.

    Science.gov (United States)

    Horton, John R; Nugent, Rebecca L; Li, Andrew; Mabuchi, Megumu Yamada; Fomenkov, Alexey; Cohen-Karni, Devora; Griggs, Rose M; Zhang, Xing; Wilson, Geoffrey G; Zheng, Yu; Xu, Shuang-yong; Cheng, Xiaodong

    2014-03-07

    The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3' to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5' to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity.

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

    Science.gov (United States)

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

    2015-10-08

    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 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.

  15. Hypoxia induces mitochondrial mutagenesis and dysfunction in inflammatory arthritis.

    LENUS (Irish Health Repository)

    Biniecka, Monika

    2012-02-01

    mitochondrial genome mutagenesis, and antioxidants significantly rescue these events.

  16. Codon cassette mutagenesis: a general method to insert or replace individual codons by using universal mutagenic cassettes.

    OpenAIRE

    Kegler-Ebo, D M; Docktor, C M; DiMaio, D

    1994-01-01

    We describe codon cassette mutagenesis, a simple method of mutagenesis that uses universal mutagenic cassettes to deposit single codons at specific sites in double-stranded DNA. A target molecule is first constructed that contains a blunt, double-strand break at the site targeted for mutagenesis. A double-stranded mutagenic codon cassette is then inserted at the target site. Each mutagenic codon cassette contains a three base pair direct terminal repeat and two head-to-head recognition sequen...

  17. 2012 Gordon Research Conference on Mutagenesis - Formal Schedule and Speaker/Poster Program

    Energy Technology Data Exchange (ETDEWEB)

    Demple, Bruce [Stony Brook Univ., NY (United States). School of Medicine

    2012-08-24

    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.

  18. Diverse responses to UV light exposure in Acinetobacter include the capacity for DNA damage-induced mutagenesis in the opportunistic pathogens Acinetobacter baumannii and Acinetobacter ursingii

    Science.gov (United States)

    Bradley, James A.; Lin, Ching-li; Elam, Tyler J.

    2012-01-01

    Error-prone and error-free DNA damage repair responses that are induced in most bacteria after exposure to various chemicals, antibiotics or radiation sources were surveyed across the genus Acinetobacter. The error-prone SOS mutagenesis response occurs when DNA damage induces a cell’s umuDC- or dinP-encoded error-prone polymerases. The model strain Acinetobacter baylyi ADP1 possesses an unusual, regulatory umuD allele (umuDAb) with an extended 5′ region and only incomplete fragments of umuC. Diverse Acinetobacter species were investigated for the presence of umuDC and their ability to conduct UV-induced mutagenesis. Unlike ADP1, most Acinetobacter strains possessed multiple umuDC loci containing either umuDAb or a umuD allele resembling that of Escherichia coli. The nearly omnipresent umuDAb allele was the ancestral umuD in Acinetobacter, with horizontal gene transfer accounting for over half of the umuDC operons. Despite multiple umuD(Ab)C operons in many strains, only three species conducted UV-induced mutagenesis: Acinetobacter baumannii, Acinetobacter ursingii and Acinetobacter beijerinckii. The type of umuDC locus or mutagenesis phenotype a strain possessed was not correlated with its error-free response of survival after UV exposure, but similar diversity was apparent. The survival of 30 Acinetobacter strains after UV treatment ranged over five orders of magnitude, with the Acinetobacter calcoaceticus–A. baumannii (Acb) complex and haemolytic strains having lower survival than non-Acb or non-haemolytic strains. These observations demonstrate that a genus can possess a range of DNA damage response mechanisms, and suggest that DNA damage-induced mutation could be an important part of the evolution of the emerging pathogens A. baumannii and A. ursingii. PMID:22117008

  19. CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme.

    Science.gov (United States)

    Michno, Jean-Michel; Wang, Xiaobo; Liu, Junqi; Curtin, Shaun J; Kono, Thomas Jy; Stupar, Robert M

    2015-01-01

    The CRISPR/Cas9 system is rapidly becoming the reagent of choice for targeted mutagenesis and gene editing in crop species. There are currently intense research efforts in the crop sciences to identify efficient CRISPR/Cas9 platforms to carry out targeted mutagenesis and gene editing projects. These efforts typically result in the incremental tweaking of various platform components including the identification of crop-specific promoters and terminators for optimal expression of the Cas9 enzyme and identification of promoters for expression of the CRISPR guide RNA. In this report, we demonstrate the development of an online web tool for fast identification of CRISPR/Cas9 target loci within soybean gene models, and generic DNA sequences. The web-tool described in this work can quickly identify a high number of potential CRISPR/Cas9 target sites, including restriction enzyme sites that can facilitate the detection of new mutations. In conjunction with the web tool, a soybean codon-optimized CRISPR/Cas9 platform was designed to direct double-stranded breaks to the targeted loci in hairy root transformed cells. The modified Cas9 enzyme was shown to successfully mutate target genes in somatic cells of 2 legume species, soybean and Medicago truncatula. These new tools may help facilitate targeted mutagenesis in legume and other plant species.

  20. An internal ribosome entry site directs translation of the 3'-gene from Pelargonium flower break virus genomic RNA: implications for infectivity.

    Science.gov (United States)

    Fernández-Miragall, Olga; Hernández, Carmen

    2011-01-01

    Pelargonium flower break virus (PFBV, genus Carmovirus) has a single-stranded positive-sense genomic RNA (gRNA) which contains five ORFs. The two 5'-proximal ORFs encode the replicases, two internal ORFs encode movement proteins, and the 3'-proximal ORF encodes a polypeptide (p37) which plays a dual role as capsid protein and as suppressor of RNA silencing. Like other members of family Tombusviridae, carmoviruses express ORFs that are not 5'-proximal from subgenomic RNAs. However, in one case, corresponding to Hisbiscus chlorotic ringspot virus, it has been reported that the 3'-proximal gene can be translated from the gRNA through an internal ribosome entry site (IRES). Here we show that PFBV also holds an IRES that mediates production of p37 from the gRNA, raising the question of whether this translation strategy may be conserved in the genus. The PFBV IRES was functional both in vitro and in vivo and either in the viral context or when inserted into synthetic bicistronic constructs. Through deletion and mutagenesis studies we have found that the IRES is contained within a 80 nt segment and have identified some structural traits that influence IRES function. Interestingly, mutations that diminish IRES activity strongly reduced the infectivity of the virus while the progress of the infection was favoured by mutations potentiating such activity. These results support the biological significance of the IRES-driven p37 translation and suggest that production of the silencing suppressor from the gRNA might allow the virus to early counteract the defence response of the host, thus facilitating pathogen multiplication and spread.

  1. An internal ribosome entry site directs translation of the 3'-gene from Pelargonium flower break virus genomic RNA: implications for infectivity.

    Directory of Open Access Journals (Sweden)

    Olga Fernández-Miragall

    Full Text Available Pelargonium flower break virus (PFBV, genus Carmovirus has a single-stranded positive-sense genomic RNA (gRNA which contains five ORFs. The two 5'-proximal ORFs encode the replicases, two internal ORFs encode movement proteins, and the 3'-proximal ORF encodes a polypeptide (p37 which plays a dual role as capsid protein and as suppressor of RNA silencing. Like other members of family Tombusviridae, carmoviruses express ORFs that are not 5'-proximal from subgenomic RNAs. However, in one case, corresponding to Hisbiscus chlorotic ringspot virus, it has been reported that the 3'-proximal gene can be translated from the gRNA through an internal ribosome entry site (IRES. Here we show that PFBV also holds an IRES that mediates production of p37 from the gRNA, raising the question of whether this translation strategy may be conserved in the genus. The PFBV IRES was functional both in vitro and in vivo and either in the viral context or when inserted into synthetic bicistronic constructs. Through deletion and mutagenesis studies we have found that the IRES is contained within a 80 nt segment and have identified some structural traits that influence IRES function. Interestingly, mutations that diminish IRES activity strongly reduced the infectivity of the virus while the progress of the infection was favoured by mutations potentiating such activity. These results support the biological significance of the IRES-driven p37 translation and suggest that production of the silencing suppressor from the gRNA might allow the virus to early counteract the defence response of the host, thus facilitating pathogen multiplication and spread.

  2. An Internal Ribosome Entry Site Directs Translation of the 3′-Gene from Pelargonium Flower Break Virus Genomic RNA: Implications for Infectivity

    Science.gov (United States)

    Fernández-Miragall, Olga; Hernández, Carmen

    2011-01-01

    Pelargonium flower break virus (PFBV, genus Carmovirus) has a single-stranded positive-sense genomic RNA (gRNA) which contains five ORFs. The two 5′-proximal ORFs encode the replicases, two internal ORFs encode movement proteins, and the 3′-proximal ORF encodes a polypeptide (p37) which plays a dual role as capsid protein and as suppressor of RNA silencing. Like other members of family Tombusviridae, carmoviruses express ORFs that are not 5′-proximal from subgenomic RNAs. However, in one case, corresponding to Hisbiscus chlorotic ringspot virus, it has been reported that the 3′-proximal gene can be translated from the gRNA through an internal ribosome entry site (IRES). Here we show that PFBV also holds an IRES that mediates production of p37 from the gRNA, raising the question of whether this translation strategy may be conserved in the genus. The PFBV IRES was functional both in vitro and in vivo and either in the viral context or when inserted into synthetic bicistronic constructs. Through deletion and mutagenesis studies we have found that the IRES is contained within a 80 nt segment and have identified some structural traits that influence IRES function. Interestingly, mutations that diminish IRES activity strongly reduced the infectivity of the virus while the progress of the infection was favoured by mutations potentiating such activity. These results support the biological significance of the IRES-driven p37 translation and suggest that production of the silencing suppressor from the gRNA might allow the virus to early counteract the defence response of the host, thus facilitating pathogen multiplication and spread. PMID:21818349

  3. Site-directed mutational analysis of structural interactions of low molecule compounds binding to the N-terminal 8 kDa domain of DNA polymerase β

    International Nuclear Information System (INIS)

    Murakami, Shizuka; Kamisuki, Shinji; Takata, Kei-ichi; Kasai, Nobuyuki; Kimura, Seisuke; Mizushina, Yoshiyuki; Ohta, Keisuke; Sugawara, Fumio; Sakaguchi, Kengo

    2006-01-01

    We previously reported the mode of inhibition of DNA polymerase β (pol. β) by long chain fatty acids and a bile acid, involving binding analyses to the N-terminal 8-kDa DNA binding domain. Here we describe a site-directed mutational analysis in which the key amino acids (L11, K35, H51, K60, L77, and T79), which are direct interaction sites in the domain, were substituted with K, A, A, A, K, and A, respectively. And their pol. β interactions with a C24-long chain fatty acid, nervonic acid (NA), and a bile acid, lithocholic acid (LCA), were investigated by gel mobility shift assay and NMR spectroscopy. In the case of K35A, there was complete loss of DNA binding activity while K60A hardly has any activity. In contrast the other mutations had no appreciable effects. Thus, K35 and K60 are key amino acid sites for binding to template DNA. The DNA binding activities of L11K, H51A, and T79A as well as the wild type were inhibited by NA to the same extent. T79A demonstrated a disturbed interaction with LCA. 1 H- 15 N HSQC NMR analysis indicated that despite their many similarities, the wild-type and the mutant proteins displayed some significant chemical shift differences. Not only were the substituted amino acid residues three-dimensionally shifted, but some amino acids which are positioned far distant from the key amino acids showed a shift. These results suggest that the interaction surface was significantly distorted with the result that LCA could not bind to the domain. These findings confirm our previous biochemical and 3D structural proposals concerning inhibition by NA and LCA

  4. Environmental Stress Induces Trinucleotide Repeat Mutagenesis in Human Cells by Alt-Nonhomologous End Joining Repair.

    Science.gov (United States)

    Chatterjee, Nimrat; Lin, Yunfu; Yotnda, Patricia; Wilson, John H

    2016-07-31

    Multiple pathways modulate the dynamic mutability of trinucleotide repeats (TNRs), which are implicated in neurodegenerative disease and evolution. Recently, we reported that environmental stresses induce TNR mutagenesis via stress responses and rereplication, with more than 50% of mutants carrying deletions or insertions-molecular signatures of DNA double-strand break repair. We now show that knockdown of alt-nonhomologous end joining (alt-NHEJ) components-XRCC1, LIG3, and PARP1-suppresses stress-induced TNR mutagenesis, in contrast to the components of homologous recombination and NHEJ, which have no effect. Thus, alt-NHEJ, which contributes to genetic mutability in cancer cells, also plays a novel role in environmental stress-induced TNR mutagenesis. Published by Elsevier Ltd.

  5. Effect of umuC mutations on targeted and untargeted ultraviolet mutagenesis in bacteriophage lambda

    International Nuclear Information System (INIS)

    Maenhaut-Michel, G.; Caillet-Fauquet, P.

    1984-01-01

    Mutagenesis of phage lambda towards clear-plaque (c + → c) results in two classes of mutants that can be distinguished genetically and morphologically. Indirect mutagenesis, i.e. mutagenesis of unirradiated phage lambdac + stimulated by the ultraviolet irradiation of the Escherichia coli host, results in mixed bursts (c/c + ) of turbid wild-type and clear=plaque mutant phages. Pure bursts of lambdac mutants are induced by irradiation of the phage genome. Irradiation of both phages and host bacteria stimulates the production of the two classes of mutant clones. It is shown that three different mutant alleles of the E. coli umuC gene only prevent the appearance of pure bursts of clear-plaque mutants, while mixed bursts are produced at least as frequently in umuC mutants as in the umuC + parent. (author)

  6. Direct Mutagenesis of Thousands of Genomic Targets using Microarray-derived Oligonucleotides

    DEFF Research Database (Denmark)

    Bonde, Mads; Kosuri, Sriram; Genee, Hans Jasper

    2015-01-01

    Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale...... operons in E. coli using this method, which we call Microarray-Oligonucleotide (MO)-MAGE. The resulting mutant library was characterized by high-throughput sequencing to show that all attempted insertions were estimated to have occurred at an average frequency of 0.02 % per loci with 0.4 average...

  7. The influence of glycerol on γ-induced mutagenesis in Salmonella typhimurium cells

    International Nuclear Information System (INIS)

    Basha, S.G.; Krasavin, E.A.; Kozubek, S.; Amirtaev, K.G.

    1990-01-01

    A study was made of the modifying effect of glycerol on the survival rate and γ-radiation-induced mutagenesis of Salmonella typhimurium cells TA98, TA100 and TA102. The DMF value, with respect to the survival rate, was 2.05-0.20. The dependence of the yield of γ-radiation-induced mutants on radiation dose was described by the curve with a maximum; the mutation frequency M(D) was well described by a gradual function M(D)=kD x . DMF values of the induced mutagenesis amounted to 2 for strains TA100 and TA102, and 1.5 for strain TA98

  8. ENU mutagenesis reveals a novel phenotype of reduced limb strength in mice lacking fibrillin 2.

    Directory of Open Access Journals (Sweden)

    Gaynor Miller

    2010-02-01

    Full Text Available Fibrillins 1 (FBN1 and 2 (FBN2 are components of microfibrils, microfilaments that are present in many connective tissues, either alone or in association with elastin. Marfan's syndrome and congenital contractural arachnodactyly (CCA result from dominant mutations in the genes FBN1 and FBN2 respectively. Patients with both conditions often present with specific muscle atrophy or weakness, yet this has not been reported in the mouse models. In the case of Fbn1, this is due to perinatal lethality of the homozygous null mice making measurements of strength difficult. In the case of Fbn2, four different mutant alleles have been described in the mouse and in all cases syndactyly was reported as the defining phenotypic feature of homozygotes.As part of a large-scale N-ethyl-N-nitrosourea (ENU mutagenesis screen, we identified a mouse mutant, Mariusz, which exhibited muscle weakness along with hindlimb syndactyly. We identified an amber nonsense mutation in Fbn2 in this mouse mutant. Examination of a previously characterised Fbn2-null mutant, Fbn2(fp, identified a similar muscle weakness phenotype. The two Fbn2 mutant alleles complement each other confirming that the weakness is the result of a lack of Fbn2 activity. Skeletal muscle from mutants proved to be abnormal with higher than average numbers of fibres with centrally placed nuclei, an indicator that there are some regenerating muscle fibres. Physiological tests indicated that the mutant muscle produces significantly less maximal force, possibly as a result of the muscles being relatively smaller in Mariusz mice.These findings indicate that Fbn2 is involved in integrity of structures required for strength in limb movement. As human patients with mutations in the fibrillin genes FBN1 and FBN2 often present with muscle weakness and atrophy as a symptom, Fbn2-null mice will be a useful model for examining this aspect of the disease process further.

  9. Discrimination of tomatoes bred by spaceflight mutagenesis using visible/near infrared spectroscopy and chemometrics.

    Science.gov (United States)

    Shao, Yongni; Xie, Chuanqi; Jiang, Linjun; Shi, Jiahui; Zhu, Jiajin; He, Yong

    2015-04-05

    Visible/near infrared spectroscopy (Vis/NIR) based on sensitive wavelengths (SWs) and chemometrics was proposed to discriminate different tomatoes bred by spaceflight mutagenesis from their leafs or fruits (green or mature). The tomato breeds were mutant M1, M2 and their parent. Partial least squares (PLS) analysis and least squares-support vector machine (LS-SVM) were implemented for calibration models. PLS analysis was implemented for calibration models with different wavebands including the visible region (400-700 nm) and the near infrared region (700-1000 nm). The best PLS models were achieved in the visible region for the leaf and green fruit samples and in the near infrared region for the mature fruit samples. Furthermore, different latent variables (4-8 LVs for leafs, 5-9 LVs for green fruits, and 4-9 LVs for mature fruits) were used as inputs of LS-SVM to develop the LV-LS-SVM models with the grid search technique and radial basis function (RBF) kernel. The optimal LV-LS-SVM models were achieved with six LVs for the leaf samples, seven LVs for green fruits, and six LVs for mature fruits, respectively, and they outperformed the PLS models. Moreover, independent component analysis (ICA) was executed to select several SWs based on loading weights. The optimal LS-SVM model was achieved with SWs of 550-560 nm, 562-574 nm, 670-680 nm and 705-71 5 nm for the leaf samples; 548-556 nm, 559-564 nm, 678-685 nm and 962-974 nm for the green fruit samples; and 712-718 nm, 720-729 nm, 968-978 nm and 820-830 nm for the mature fruit samples. All of them had better performance than PLS and LV-LS-SVM, with the parameters of correlation coefficient (rp), root mean square error of prediction (RMSEP) and bias of 0.9792, 0.2632 and 0.0901 based on leaf discrimination, 0.9837, 0.2783 and 0.1758 based on green fruit discrimination, 0.9804, 0.2215 and -0.0035 based on mature fruit discrimination, respectively. The overall results indicated that ICA was an effective way for the

  10. Characterization of antibodies in single-chain format against the E7 oncoprotein of the Human papillomavirus type 16 and their improvement by mutagenesis

    Directory of Open Access Journals (Sweden)

    Accardi Luisa

    2007-01-01

    Full Text Available Abstract Background Human papillomaviruses (HPV are the etiological agents of cervical cancer. The viral E7 protein plays a crucial role in viral oncogenesis. Many strategies have been explored to block the E7 oncoprotein activity. The single-chain variable antibody fragments (scFvs are valuable tools in cancer immunotherapy and can be used as "intracellular antibodies" to knock out specific protein functions. For both in vivo and in vitro employment, the scFv intrinsic solubility and stability are important to achieve long-lasting effects. Here we report the characterization in terms of reactivity, solubility and thermal stability of three anti-HPV16 E7 scFvs. We have also analysed the scFv43 sequence with the aim of improving stability and then activity of the antibody, previously shown to have antiproliferative activity when expressed in HPV16-positive cells. Methods The three anti-HPV16 E7 scFv 32, 43 51 were selected from the ETH-2 "phage-display" library. Thermal stability was evaluated with ELISA by determining the residual activity of each purified scFv against the recombinant HPV16 E7, after incubation in the presence of human seroalbumine for different time-intervals at different temperatures. Sequence analysis of the scFvs was performed with BLAST and CLUSTALL programs. The scFv43 aminoacid changes were reverted back to the consensus sequence from the immunoglobuline database by site-directed mutagenesis. ScFv solubility was evaluated with Western blotting by determining their relative amounts in the soluble and insoluble fractions of both prokaryotic and eukaryotic systems. Results ScFv51 was the most thermally stable scFv considered. Sequence analysis of the most reactive scFv43 has evidenced 2 amino acid changes possibly involved in molecule stability, in the VH and VL CDR3 regions respectively. By mutagenesis, two novel scFv43-derived scFvs were obtained, scFv43 M1 and M2. ScFv43 M2 showed to have improved thermal stability and

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans▿

    Science.gov (United States)

    Xie, Zhoujie; Okinaga, Toshinori; Qi, Fengxia; Zhang, Zhijun; Merritt, Justin

    2011-01-01

    Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed that utilize either temperature-sensitive plasmids or counterselectable suicide vectors containing both positive- and negative-selection markers. For many species, these techniques are not especially useful due to difficulties of cloning with Escherichia coli and/or a lack of functional negative-selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable to a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows extremely quick assembly and eliminates the requirement for either temperature-sensitive replicons or suicide vectors. As a proof of principle, we used Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild-type S. mutans strains, we further demonstrated that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches. PMID:21948849

  13. Cloning-independent and counterselectable markerless mutagenesis system in Streptococcus mutans.

    Science.gov (United States)

    Xie, Zhoujie; Okinaga, Toshinori; Qi, Fengxia; Zhang, Zhijun; Merritt, Justin

    2011-11-01

    Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed that utilize either temperature-sensitive plasmids or counterselectable suicide vectors containing both positive- and negative-selection markers. For many species, these techniques are not especially useful due to difficulties of cloning with Escherichia coli and/or a lack of functional negative-selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable to a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows extremely quick assembly and eliminates the requirement for either temperature-sensitive replicons or suicide vectors. As a proof of principle, we used Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild-type S. mutans strains, we further demonstrated that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches.

  14. Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

    Czech Academy of Sciences Publication Activity Database

    Hoffman, Hillary Elizabeth; Jirásková, Jana; Zvelebil, M.; Konvalinka, Jan

    2010-01-01

    Roč. 75, č. 1 (2010), s. 59-79 ISSN 0010-0765 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : D-serine * serine racemase * random mutagenesis Subject RIV: CE - Biochemistry Impact factor: 0.853, year: 2010

  15. DNA polymerase III of Escherichia coli is required for UV and ethyl methanesulfonate mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Hagensee, M.E.; Timme, T.L.; Bryan, S.K.; Moses, R.E.

    1987-06-01

    Strains of Escherichia coli possessing the pcbA1 mutation, a functional DNA polymerase I, and a temperature-sensitive mutation in DNA polymerase III can survive at the restrictive temperature (43 degrees C) for DNA polymerase III. The mutation rate of the bacterial genome of such strains after exposure to either UV light or ethyl methanesulfonate was measured by its rifampicin resistance or amino acid requirements. In addition, Weigle mutagenesis of preirradiated lambda phage was also measured. In all cases, no increase in mutagenesis was noted at the restrictive temperature for DNA polymerase III. Introduction of a cloned DNA polymerase III gene returned the mutation rate of the bacterial genome as well as the Weigle mutagenesis to normal at 43 degrees C. Using a recA-lacZ fusion, the SOS response after UV irradiation was measured and found to be normal at the restrictive and permissive temperature for DNA polymerase III, as was induction of lambda prophage. Recombination was also normal at either temperature. Our studies demonstrate that a functional DNA polymerase III is strictly required for mutagenesis at a step other than SOS induction.

  16. Mutagenesis and functional studies with succinate dehydrogenase inhibitors in the wheat pathogen Mycosphaerella graminicola.

    Directory of Open Access Journals (Sweden)

    Gabriel Scalliet

    Full Text Available A range of novel carboxamide fungicides, inhibitors of the succinate dehydrogenase enzyme (SDH, EC 1.3.5.1 is currently being introduced to the crop protection market. The aim of this study was to explore the impact of structurally distinct carboxamides on target site resistance development and to assess possible impact on fitness. We used a UV mutagenesis approach in Mycosphaerella graminicola, a key pathogen of wheat to compare the nature, frequencies and impact of target mutations towards five subclasses of carboxamides. From this screen we identified 27 amino acid substitutions occurring at 18 different positions on the 3 subunits constituting the ubiquinone binding (Qp site of the enzyme. The nature of substitutions and cross resistance profiles indicated significant differences in the binding interaction to the enzyme across the different inhibitors. Pharmacophore elucidation followed by docking studies in a tridimensional SDH model allowed us to propose rational hypotheses explaining some of the differential behaviors for the first time. Interestingly all the characterized substitutions had a negative impact on enzyme efficiency, however very low levels of enzyme activity appeared to be sufficient for cell survival. In order to explore the impact of mutations on pathogen fitness in vivo and in planta, homologous recombinants were generated for a selection of mutation types. In vivo, in contrast to previous studies performed in yeast and other organisms, SDH mutations did not result in a major increase of reactive oxygen species levels and did not display any significant fitness penalty. However, a number of Qp site mutations affecting enzyme efficiency were shown to have a biological impact in planta.Using the combined approaches described here, we have significantly improved our understanding of possible resistance mechanisms to carboxamides and performed preliminary fitness penalty assessment in an economically important plant pathogen

  17. Effectiveness of gamma-ray chronic irradiation on in vitro mutagenesis in crops

    International Nuclear Information System (INIS)

    Shigeki Nagatomi

    2002-01-01

    Effects of chronic or acute irradiations were compared using in vitro culture on inducing the mutation in model crops. In chrysanthemum, combined method with irradiation and in vitro culture can solve the problem of chimera formation in induced mutants, and provided 10 times greater mutation frequency than usual plant irradiation. The chronic culture method showed the widest color spectrum, whereas, the acute culture indicated a relatively low mutation rate and a very limited flower color spectrum in chrysanthemum. Flower color mutation of the regenerators could be induced more from petals and buds than from leaves. These facts are supposed that the gene loci fully expressed on floral organs may be unstable for mutation by mutagenesis or culture. It may be likely to control a direction of desired mutation on using explants with specific gene loci activated. In sugarcane, the chronic culture method extended quantitative characteristics of regenerated clonal lines toward not only the negative but positive direction. On the other hand, the acute culture method showed lower quantitative mutation as the irradiation dose rose. In chronic irradiation, regenerated mutant lines in sugarcane indicate generally little decrease in chromosome number and wider variations with relatively less damage. In acute irradiation, regenerated mutant lines show remarkable decrease of chromosome numbers in sugarcane mutant lines as the irradiation dose rose. There is close positive correlation between chromosome number and biomass of each mutant line. The chromosome number estimation is a proper indicator to monitor damage of adopted irradiation methods. Possible reason why the chronic culture methods indicate higher frequency and wider spectrum on mutation is demonstrated. . Problems solved and prospect of chronic irradiation and in vitro techniques are discussed. (Author)

  18. Modelling of the binding site of the human m1 muscarinic receptor: Experimental validation and refinement

    Science.gov (United States)

    Bourdon, Hélène; Trumpp-Kallmeyer, Susanne; Schreuder, Herman; Hoflack, , Jan; Hibert, Marcel; Wermuth, Camille-Georges

    1997-07-01

    Our model of the human m1 muscarinic receptor has been refined on the basis of the recently published projection map of bovine rhodopsin. The refined model has a slightly different helix arrangement, which reveals the presence of an extra hydrophobic pocket located between helices 3, 4 and 5. The interaction of series of agonists and antagonists with the m1 muscarinic receptor has been studied experimentally by site-directed mutagenesis. In order to account for the observed results, three-dimensional models of m1 ligands docked in the target receptor are proposed. Qualitatively, the obtained models are in good agreement with the experimental observations. Agonists and partial agonists have a relatively small size. They can bind to the same region of the receptor using, however, different anchoring receptor residues. Antagonists are usually larger molecules, filling almost completely the same pocket as agonists. They can usually produce much stronger interactions with aromatic residues. Experimental data combined with molecular modelling studies highlight how subtle and diverse receptor-ligand interactions could be.

  19. A study on the effect of surface lysine to arginine mutagenesis on protein stability and structure using green fluorescent protein.

    Science.gov (United States)

    Sokalingam, Sriram; Raghunathan, Govindan; Soundrarajan, Nagasundarapandian; Lee, Sun-Gu

    2012-01-01

    Two positively charged basic amino acids, arginine and lysine, are mostly exposed to protein surface, and play important roles in protein stability by forming electrostatic interactions. In particular, the guanidinium group of arginine allows interactions in three possible directions, which enables arginine to form a larger number of electrostatic interactions compared to lysine. The higher pKa of the basic residue in arginine may also generate more stable ionic interactions than lysine. This paper reports an investigation whether the advantageous properties of arginine over lysine can be utilized to enhance protein stability. A variant of green fluorescent protein (GFP) was created by mutating the maximum possible number of lysine residues on the surface to arginines while retaining the activity. When the stability of the variant was examined under a range of denaturing conditions, the variant was relatively more stable compared to control GFP in the presence of chemical denaturants such as urea, alkaline pH and ionic detergents, but the thermal stability of the protein was not changed. The modeled structure of the variant indicated putative new salt bridges and hydrogen bond interactions that help improve the rigidity of the protein against different chemical denaturants. Structural analyses of the electrostatic interactions also confirmed that the geometric properties of the guanidinium group in arginine had such effects. On the other hand, the altered electrostatic interactions induced by the mutagenesis of surface lysines to arginines adversely affected protein folding, which decreased the productivity of the functional form of the variant. These results suggest that the surface lysine mutagenesis to arginines can be considered one of the parameters in protein stability engineering.

  20. Topoisomerase II-mediated DNA cleavage and mutagenesis activated by nitric oxide underlie the inflammation-associated tumorigenesis.

    Science.gov (United States)

    Yang, Yu-Chen; Chou, Han-Yi E; Shen, Tang-Long; Chang, Wei-Jer; Tai, Pei-Han; Li, Tsai-Kun

    2013-04-01

    Both cancer-suppressing and cancer-promoting properties of reactive nitrogen and oxygen species (RNOS) have been suggested to play a role in tumor pathology, particularly those activities associated with chronic inflammation. Here, we address the impact of nitric oxide (NO) on the induction of DNA damage and genome instability with a specific focus on the involvement of topoisomerase II (TOP2). We also investigate the contribution of NO to the formation of skin melanoma in mice. Similar to the TOP2-targeting drug, etoposide (VP-16), the NO-donor, S-nitrosoglutathione (GSNO), induces skin melanomas formation in 7,12-dimethyl- benz[a]anthracene (DMBA)-initiated mice. To explore the mechanism(s) underlying this NO-induced tumorigenesis, we use a co-culture model system to demonstrate that inflamed macrophages with inducible NO synthase (iNOS) expression cause γ-H2AX activation, p53 phosphorylation, and chromosome DNA breaks in the target cells. Inhibitor experiments revealed that NO and TOP2 isozymes are responsible for the above described cellular phenotypes. Notably, NO, unlike VP-16, preferentially induces the formation of TOP2β cleavable complexes (TOP2βcc) in cells. Moreover, GSNO induced TOP2-dependent DNA sequence rearrangements and cytotoxicity. Furthermore, the incidences of GSNO- and VP-16-induced skin melanomas were also observed to be lower in the skin-specific top2β-knockout mice. Our results suggest that TOP2 isozymes contribute to NO-induced mutagenesis and subsequent cancer development during chronic inflammation. We provide the first experimental evidence for the functional role of TOP2 in NO-caused DNA damage, mutagenesis, and carcinogenesis. Notably, these studies contribute to our molecular understanding of the cancer-promoting actions of RNOS during chronic inflammation.

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

    Directory of Open Access Journals (Sweden)

    Emily J Capra

    2010-11-01

    Full Text Available 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.

  2. Casting epPCR (cepPCR): A simple random mutagenesis method to generate high quality mutant libraries.

    Science.gov (United States)

    Yang, Jianhua; Ruff, Anna J; Arlt, Marcus; Schwaneberg, Ulrich

    2017-09-01

    During the last decade, directed evolution has become a standard protein engineering strategy to reengineer proteins for industrial applications under high stress conditions (e.g., high temperature, extreme pH, ionic liquids, or organic solvents). The most commonly employed method for diversity generation to improve biocatalysts for these properties is random mutagenesis by error-prone polymerase chain reaction (epPCR). However, recent reports show that epPCR often fails to produce >70% of beneficial positions/amino acid exchanges which improve enzyme properties such as organic solvent or ionic liquid resistance. In this report, bsla (543 bp, small lipase gene from Bacillus subtilis) was divided into three fragments (147, 192, 204 bp). Each fragment was subjected to an epPCR with a high mutation load (22, 31, and 33 mutations per kb) in order to increase the number of identified beneficial positions while maintaining a fraction of active population which can efficiently be screened in agar plate or microtiter plate format. The use of this "casting epPCR" process termed as (cepPCR), doubles the number of identified beneficial positions (from 14% to 29%), when compared to standard epPCR for the BSLA enzyme model. A further increase to 39% of beneficial positions is obtainable through combination of cepPCR with the transversion biased sequence saturation mutagenesis (SeSaM) method. Furthermore, sequencing of up to 600 mutations per fragment provided valuable insights into the correlation of total throughput and number of identified beneficial positions as well as how an efficient balance of screening efforts to obtainable results can be achieved in directed evolution campaigns. Biotechnol. Bioeng. 2017;114: 1921-1927. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    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…

  4. Molecular dynamics simulations and structure-guided mutagenesis provide insight into the architecture of the catalytic core of the ectoine hydroxylase.

    Science.gov (United States)

    Widderich, Nils; Pittelkow, Marco; Höppner, Astrid; Mulnaes, Daniel; Buckel, Wolfgang; Gohlke, Holger; Smits, Sander H J; Bremer, Erhard

    2014-02-06

    Many bacteria amass compatible solutes to fend-off the detrimental effects of high osmolarity on cellular physiology and water content. These solutes also function as stabilizers of macromolecules, a property for which they are referred to as chemical chaperones. The tetrahydropyrimidine ectoine is such a compatible solute and is widely synthesized by members of the Bacteria. Many ectoine producers also synthesize the stress protectant 5-hydroxyectoine from the precursor ectoine, a process that is catalyzed by the ectoine hydroxylase (EctD). The EctD enzyme is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. A crystal structure of the EctD protein from the moderate halophile Virgibacillus salexigens has previously been reported and revealed the coordination of the iron catalyst, but it lacked the substrate ectoine and the co-substrate 2-oxoglutarate. Here we used this crystal structure as a template to assess the likely positioning of the ectoine and 2-oxoglutarate ligands within the active site by structural comparison, molecular dynamics simulations, and site-directed mutagenesis. Collectively, these approaches suggest the positioning of the iron, ectoine, and 2-oxoglutarate ligands in close proximity to each other and with a spatial orientation that will allow the region-selective and stereo-specific hydroxylation of (4S)-ectoine to (4S,5S)-5-hydroxyectoine. Our study thus provides a view into the catalytic core of the ectoine hydroxylase and suggests an intricate network of interactions between the three ligands and evolutionarily highly conserved residues in members of the EctD protein family. © 2013.

  5. D181A Site-Mutagenesis Enhances Both the Hydrolyzing and Transfructosylating Activities of BmSUC1, a Novel β-Fructofuranosidase in the Silkworm Bombyx mori

    Directory of Open Access Journals (Sweden)

    Quan Gan

    2018-02-01

    Full Text Available β-fructofuranosidase (β-FFase belongs to the glycosyl-hydrolase family 32 (GH32, which can catalyze both the release of β-fructose from β-d-fructofuranoside substrates to hydrolyze sucrose and the synthesis of short-chain fructooligosaccharide (FOS. BmSuc1 has been cloned and identified from the silkworm Bombyx mori as a first animal type of β-FFase encoding gene. It was hypothesized that BmSUC1 plays an important role in the silkworm-mulberry adaptation system. However, there is little information about the enzymatic core sites of BmSUC1. In this study, we mutated three amino acid residues (D63, D181, and E234 that represent important conserved motifs for β-FFase activity in GH32 to alanine respectively by using site-directed mutagenesis. Recombinant proteins of three mutants and wild type BmSUC1 were obtained by using a Bac-to-Bac/BmNPV expression system and BmN cells. Enzymatic activity, kinetic properties, and substrate specificity of the four proteins were analyzed. High Performance Liquid Chromatography (HPLC was used to compare the hydrolyzing and transfructosylating activities between D181A and wtBmSUC1. Our results revealed that the D63A and E234A mutations lost activity, suggesting that D63 and E234 are key amino acid residues for BmSUC1 to function as an enzyme. The D181A mutation significantly enhanced both hydrolyzing and transfructosylating activities of BmSUC1, indicating that D181 may not be directly involved in catalyzation. The results provide insight into the chemical catalyzation mechanism of BmSUC1 in B. mori. Up-regulated transfructosylating activity of BmSUC1 could provide new ideas for using B. mori β-FFase to produce functional FOS.

  6. Enzyme kinetics, inhibitors, mutagenesis and electron paramagnetic resonance analysis of dual-affinity nitrate reductase in unicellular N(2)-fixing cyanobacterium Cyanothece sp. PCC 8801.

    Science.gov (United States)

    Wang, Tung-Hei; Chen, Yung-Han; Huang, Jine-Yung; Liu, Kang-Cheng; Ke, Shyue-Chu; Chu, Hsiu-An

    2011-11-01

    The assimilatory nitrate reductase (NarB) of N(2)-fixing cyanobacterium Cyanothece sp. PCC 8801 is a monomeric enzyme with dual affinity for substrate nitrate. We purified the recombinant NarB of Cyanothece sp. PCC 8801 and further investigated it by enzyme kinetics analysis, site-directed mutagenesis, inhibitor kinetics analysis, and electron paramagnetic resonance (EPR) spectroscopy. The NarB showed 2 kinetic regimes at pH 10.5 or 8 and electron-donor conditions methyl viologen or ferredoxin (Fd). Fd-dependent NR assay revealed NarB with very high affinity for nitrate (K(m)1, ∼1μM; K(m)2, ∼270μM). Metal analysis and EPR results showed that NarB contains a Mo cofactor and a [4Fe-4S] cluster. In addition, the R352A mutation on the proposed nitrate-binding site of NarB greatly altered both high- and low-affinity kinetic components. Furthermore, the effect of azide on the NarB of Cyanothece sp. PCC 8801 was more complex than that on the NarB of Synechococcus sp. PCC 7942 with its single kinetic regime. With 1mM azide, the kinetics of the wild-type NarB was transformed from 2 kinetic regimes to hyperbolic kinetics, and its activity was enhanced significantly under medium nitrate concentrations. Moreover, EPR results also suggested a structural difference between the two NarBs. Taken together, our results show that the NarB of Cyanothece sp. PCC 8801 contains only a single Mo-catalytic center, and we rule out that the enzyme has 2 independent, distinct catalytic sites. In addition, the NarB of Cyanothece sp. PCC 8801 may have a regulatory nitrate-binding site. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  7. Detección de una mutación no estándar en el Proto-oncogen RET por mutagénesis dirigida Detection of a non-standard mutation in the ret protoncogene by site directed mutagenesis

    Directory of Open Access Journals (Sweden)

    Sebastián Real

    2005-03-01

    Full Text Available El síndrome de MEN2A es una enfermedad autosómica dominante que se caracteriza por el desarrollo de cáncer medular de tiroides, feocromocitoma e hiperplasia de paratiroides. Mutaciones en el ret proto-oncogén se asocian con MEN2A, con una penetrancia cercana al 100%. El gen se encuentra en el cromosoma 10q11.2 y codifica para una proteína transmembrana con función de receptor del tipo tirosina quinasa. Mutaciones que afectan el dominio extracelular de la proteína estimulan la dimerización espontánea del receptor y un aumento de la actividad de tirosina quinasa basal. El codón 634 codifica para una cisteína, y es considerado un sitio hot-spot por encontrarse mutado en el 85% de las familias con MEN2A. Para este sitio, nuestro grupo desarrolló en 2002 una metodología de detección indirecta y económica. Ante una familia sospechada de MEN2A, se aplicó esta estrategia, que reveló un codón 634 sano. Por posterior secuenciación se confirmó que el paciente índice portaba una mutación en el codón 611. Se desarrolló una nueva estrategia familia-específica por PCR mutagénica, que permitió diagnosticar en nuestro país a todos los integrantes de la familia con costos accesibles. Un niño en el cual se halló la mutación, fue tiroidectomizado preventivamente, y a la fecha goza de buena salud. De esta manera, combinando la estrategia de detección de mutaciones en el sitio hot-spot y un posterior diseño de otra metodología familia-específica se pudo diagnosticar e intervenir preventivamente a la familia, sin enviar todas las muestras al extranjero.MEN2A is an autosomic dominant disease, characterized by medullary thyroid cancer, pheochromocytoma and parathyroid hyperplasia. Mutations in the ret proto-oncogene are associated with this disease, with almost 100% of pennetrance. The gene, situated on chromosome 10q11.2, codes for a transmembrane protein with a tirosinkinase-like receptor function. Mutations that affect its extracellular domain, stimulate spontaneous homodimerization and elevate the basal tirosinkinase activity. The codon 634 of the gene is considered a hot-spot site, since it is mutated in 85% of the MEN2A families. Our group developed in 2002 an indirect and costless strategy to detect alterations in this site. We present a family suspected of having MEN2A. We applied our PCR based indirect strategy on the DNA of the index patient and found that there was no mutation in that site. Posterior sequencing of exon 10 and 11 confirmed that the mutation affecting this family was in codon 611. Thus, we developed a new costless family-specific strategy based on mutagenic PCR and enzymatic cuts to diagnose all the family members. A seven-year old boy with this mutation was preventively thyroidectomized. In this way, combining the indirect methodology for codon 634 previously developed by our group, and a posterior family-specific mutation detection strategy, we were able to diagnose and intervene presymptomaticly the family members, avoiding sending all the samples to foreign centers.

  8. Analysis of Active-Site Amino-Acid Residues of Human Serum Paraoxonase Using Competitive Substrates

    National Research Council Canada - National Science Library

    Yeung, David T; Lenz, David E; Cerasoli, Douglas M

    2005-01-01

    ...(s) or its catalytic mechanism. Through site-directed mutagenesis studies, designed from a DFPase-like homology model, and from a crystal structure of a hybrid PONl molecule, amino-acid residues essential for enzyme function...

  9. ENU Mutagenesis Screen to Establish Motor Phenotypes in Wild-Type Mice and Modifiers of a Pre-Existing Motor Phenotype in Tau Mutant Mice

    Directory of Open Access Journals (Sweden)

    Xin Liu

    2011-01-01

    Full Text Available Modifier screening is a powerful genetic tool. While not widely used in the vertebrate system, we applied these tools to transgenic mouse strains that recapitulate key aspects of Alzheimer’s disease (AD, such as tau-expressing mice. These are characterized by a robust pathology including both motor and memory impairment. The phenotype can be modulated by ENU mutagenesis, which results in novel mutant mouse strains and allows identifying the underlying gene/mutation. Here we discuss this strategy in detail. We firstly obtained pedigrees that modify the tau-related motor phenotype, with mapping ongoing. We further obtained transgene-independent motor pedigrees: (i hyperactive, circling ENU 37 mice with a causal mutation in the Tbx1 gene—the complete knock-out of Tbx1 models DiGeorge Syndrome; (ii ENU12/301 mice that show sudden jerky movements and tremor constantly; they have a causal mutation in the Kcnq1 gene, modelling aspects of the Romano-Ward and Jervell and Lange-Nielsen syndromes; and (iii ENU16/069 mice with tremor and hypermetric gait that have a causal mutation in the Mpz (Myelin Protein Zero gene, modelling Charcot-Marie-Tooth disease type 1 (CMT1B. Together, we provide evidence for a real potential of an ENU mutagenesis to dissect motor functions in wild-type and tau mutant mice.

  10. Structural Probing and Molecular Modeling of the A₃ Adenosine Receptor: A Focus on Agonist Binding.

    Science.gov (United States)

    Ciancetta, Antonella; Jacobson, Kenneth A

    2017-03-11

    Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR) subtypes, termed A₁, A 2A , A 2B and A₃, which belong to the G protein-coupled receptor (GPCR) superfamily. The human A₃AR (hA₃AR) subtype is implicated in several cytoprotective functions. Therefore, hA₃AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure-activity relationships (SARs) of newly emerged A₃AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM) data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A₃AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates.

  11. Structural Probing and Molecular Modeling of the A3 Adenosine Receptor: A Focus on Agonist Binding

    Science.gov (United States)

    Ciancetta, Antonella; Jacobson, Kenneth A.

    2017-01-01

    Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR) subtypes, termed A1, A2A, A2B and A3, which belong to the G protein-coupled receptor (GPCR) superfamily. The human A3AR (hA3AR) subtype is implicated in several cytoprotective functions. Therefore, hA3AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure-activity relationships (SARs) of newly emerged A3AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM) data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A3AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates. PMID:28287473

  12. Structural Probing and Molecular Modeling of the A3 Adenosine Receptor: A Focus on Agonist Binding

    Directory of Open Access Journals (Sweden)

    Antonella Ciancetta

    2017-03-01

    Full Text Available Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR subtypes, termed A1, A2A, A2B and A3, which belong to the G protein-coupled receptor (GPCR superfamily. The human A3AR (hA3AR subtype is implicated in several cytoprotective functions. Therefore, hA3AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure–activity relationships (SARs of newly emerged A3AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A3AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates.

  13. Structural Modelling and Structure-Function Analysis of Zymomonas mobilis Levansucrase

    Directory of Open Access Journals (Sweden)

    Bahar BAKAR

    2017-03-01

    Full Text Available Levansucrases are bacterial enzymes which produce fructan polymers from sucrose via hydrolysis and transfructosylation activities. These polymers; levan and fructooligosaccharides are valuable for food and pharmaceutical industries. Levansucrases from Gram-positive bacteria such as Bacillus subtilis tend to produce levan, while those from Gram-negative bacteria preferentially produce fructooligosaccharides. Zymomonas mobilis is an efficient levansucrase producer and its extracellular levansucrase can produce both fructooligosaccharides and levan depending on the reaction parameters. In this study, the structure of Z. mobilis levansucrase was modeled in order to help to understand the structure-function relationship of the enzyme. Furthermore, amino acids previously reported to be important for levansucrase activity were mapped on the model. The structural model presents a five-bladed propeller with a deep, negatively charged central pocket, similar to other bacterial levansucrases. Mapping showed that amino acids which previously reported to affect fructan length are located on the periphery of the structure covering the active site central pocket. Thus it is showed that, for the first time, that hydrolysis and transfructosylation reactions are catalyzed on different parts of Z. mobilis levansucrase structure. The structural location of the critical amino acids will pave the way to identify other residues which control fructan length by site directed mutagenesis without altering the overall fold of the enzyme.

  14. Altered lipid accumulation in Nannochloropsis salina CCAP849/3 following EMS and UV induced mutagenesis

    Directory of Open Access Journals (Sweden)

    T.A. Beacham

    2015-09-01

    Full Text Available 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.

  15. Altered lipid accumulation in Nannochloropsis salina CCAP849/3 following EMS and UV induced mutagenesis.

    Science.gov (United States)

    Beacham, T A; Macia, V Mora; Rooks, P; White, D A; Ali, S T

    2015-09-01

    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.

  16. The mutagenesis and breeding of high productive strains of streptomyces jingyangensis '5406'

    International Nuclear Information System (INIS)

    Qi Hongyan; Yin Xinyun

    1988-03-01

    The purpose of these experiments is to explore the mutagenesis rhythm and breed high productive strains of actinomycete '5406'. The single colony agar pieces of strain F 358 were treated with fast neutron and 60 Co-γ ray irradiation Two mutants have been selected from 20025 treated single colonies. The output of cytokinins from them is higher than from strain F 358 . The original strain 'Mu-Tan-al' rejuvenated by freezing was treated with several physical and chemical mutagens. The mutagenesis rhythm has been summed up tentatively. Eight mutants obtained from 93014 treated single colonies produced more '5406' antibiotics than that of strain 'Mu-Tan-al,. The effect of mutant 'N2-10-Ra3' was the best

  17. Mutagenesis of the bacterial RNA polymerase alpha subunit for improvement of complex phenotypes.

    Science.gov (United States)

    Klein-Marcuschamer, Daniel; Santos, Christine Nicole S; Yu, Huimin; Stephanopoulos, Gregory

    2009-05-01

    Combinatorial or random methods for strain engineering have been extensively used for the improvement of multigenic phenotypes and other traits for which the underlying mechanism is not fully understood. Although the preferred method has traditionally been mutagenesis and selection, our laboratory has successfully used mutant transcription factors, which direct the RNA polymerase (RNAP) during transcription, to engineer complex phenotypes in microbial cells. Here, we show that it is also possible to impart new phenotypes by altering the RNAP core enzyme itself, in particular through mutagenesis of the alpha subunit of the bacterial polymerase. We present the use of this tool for improving tolerance of Escherichia coli to butanol and other solvents and for increasing the titers of two commercially relevant products, L-tyrosine and hyaluronic acid. In addition, we explore the underlying physiological changes that give rise to the solvent-tolerant mutant.

  18. Mutagenesis of the somaclones in vitro of cut roses by 60Co γ-rays irradiation

    International Nuclear Information System (INIS)

    Qu Suping; Su Yan; Wang Lihua; Tang Kaixue; Wang Jihua; Zhang Hao

    2009-01-01

    Mutagenesis of cut rose in vitro irradiated by 60 Co γ-rays was studied. The callus of leaves and regenerations adventitious bud was used as the explants for mutagenesis. Effect of 60 Co γ-rays irradiation on the callus's regeneration rate, adventitious bud's multiplication rate and vegetal status were studied. The results showed that the regeneration frequency of callus was decreased by 60 Co γ-rays irradiation. The regenerations adventitious bud was the better experimental materials compared with the callus of leaves. The lethal dose was 122 Gy and the semi-lethal dose was 76 Gy according to the regression equation. The appropriate dose on adventitious bud by irradiation rays was 50-60 Gy. (authors)

  19. Regulation of mutagenesis by exogenous biological factors in the eukaryotic cell systems

    Directory of Open Access Journals (Sweden)

    Lukash L. L.

    2013-07-01

    Full Text Available The representations of the mutations and the nature of spontaneous mutation process and mutagenesis induced by exogenous oncoviruses, DNAs and proteins-mitogens are analysed. Exogenous biological factors induce DNA damages in regulatory-informational way, acting on the cellular systems for maintenance of genetical stability. Molecular mechanisms are the same as at spontaneous mutagenesis but they are realized with the participation of alien genetical material. Among biological mutagens, the oncoviruses and mobile genetic elements (MGEs are distinguished as the strongest destabilizing factors which direct tumor transformation of somatic mammalian cells. Genetical reprogramming or changing the programs of gene expression at the differentiation of stem and progenitor cells under growth factors and citokines is probably followed by mutations and recombinations as well.

  20. Cell-mediated mutagenesis and cell transformation of mammalian cells by chemical carcinogens

    International Nuclear Information System (INIS)

    Huberman, E.; Langenbach, R.

    1977-01-01

    We have developed a cell-mediated mutagenesis assay in which cells with the appropriate markers for mutagenesis are co-cultivated with either lethally irradiated rodent embryonic cells that can metabolize carcinogenic hydrocarbons or with primary rat liver cells that can metabolize chemicals carcinogenic to the liver. During co-cultivation, the reactive metabolites of the procarcinogen appear to be transmitted to the mutable cells and induce mutations in them. Assays of this type make it possible to demonstrate a relationship between carcinogenic potency of the chemicals and their ability to induce mutations in mammalian cells. In addition, by simultaneously comparing the frequencies of transformation and mutation induced in normal diploid hamster cells by benzo(a)pyrene (BP) and one of its metabolites, it is possible to estimate the genetic target size for cell transformation in vitro

  1. Mutagenesis of Jatropha curcas - Exploring new traits in the breeding of a biofuel plant

    International Nuclear Information System (INIS)

    Azhar Mohamad; Sobri Hussein; Abdul Rahim Harun

    2010-01-01

    Mutagenesis in plant species is considered effective in recovering and producing useful mutants as it leads to a high degree of chimerism and produces high degree of somaclonal variations for further selection in breeding programmes. Jatropha curcas is a species with many attributes and considerable potential, especially as bio diesel. Narrow genetic background of Jatropha spp. gives less selection to growers for better quality plant materials. In this study, a new method through nuclear technology was used to increase the genetic variability of Jatropha towards novel superior potential mutant lines. The objective of the study is to generate new mutant varieties of Jatropha curcas through the mutagenesis approach in getting new sustainable mutants for high oil yield and improved plant characteristics. Seeds of a Jatropha cultivar were from selected materials from Lembaga Kenaf and Tembakau Negara, Kelantan. Radiosensitivity test was done by irradiating a total of each 60 seeds at multiple doses (0 Gy, 20 Gy, 40 Gy, 60 Gy, 80 Gy, 100 Gy, 200 Gy, 300 Gy, 400 Gy, 600 Gy and 700 Gy). After getting the LD 50 , three doses i.e. 250 Gy, 300 Gy and 350 Gy were selected for mutagenesis, where a total of 1000 seeds were exposed to gamma radiation. The seeds were hardened and field planted at close distance of 1 m x 1 m. Pruning was conducted three times at two months interval prior to screening for early flowering, short stature and high branching mutant lines. Radiosensitivity of seeds to acute gamma irradiation revealed that the LD 50 was at 320 Gy. At nursery stage, somatic mutations related to chlorophyll changes were observed on leaves with certain shapes. Screening of Jatropha via seed mutagenesis bore 6 early flowering mutants, 7 dwarf mutants and, 17 high branching plants. In narrowing the mutant lines, cuttings from each selected trait were collected and re-planted for further evaluation. (author)

  2. The contribution of Nth and Nei DNA glycosylases to mutagenesis in Mycobacterium smegmatis.

    Science.gov (United States)

    Moolla, Nabiela; Goosens, Vivianne J; Kana, Bavesh D; Gordhan, Bhavna G

    2014-01-01

    The increased prevalence of drug resistant strains of Mycobacterium tuberculosis (Mtb) indicates that significant mutagenesis occurs during tuberculosis disease in humans. DNA damage by host-derived reactive oxygen/nitrogen species is hypothesized to be critical for the mutagenic process in Mtb thus, highlighting an important role for DNA repair enzymes in maintenance of genome fidelity. Formamidopyrimidine (Fpg/MutM/Fapy) and EndonucleaseVIII (Nei) constitute the Fpg/Nei family of DNA glycosylases and together with EndonucleaseIII (Nth) are central to the base excision repair pathway in bacteria. In this study we assess the contribution of Nei and Nth DNA repair enzymes in Mycobacterium smegmatis (Msm), which retains a single nth homologue and duplications of the Fpg (fpg1 and fpg2) and Nei (nei1 and nei2) homologues. Using an Escherichia coli nth deletion mutant, we confirm the functionality of the mycobacterial nth gene in the base excision repair pathway. Msm mutants lacking nei1, nei2 and nth individually or in combination did not display aberrant growth in broth culture. Deletion of nth individually results in increased UV-induced mutagenesis and combinatorial deletion with the nei homologues results in reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin. Deletion of nth together with the fpg homolgues did not result in any growth/survival defects or changes in mutation rate. Furthermore, no differential emergence of the common rifampicin resistance conferring genotypes were noted. Collectively, these data confirm a role for Nth in base excision repair in mycobacteria and further highlight a novel interplay between the Nth and Nei homologues in spontaneous mutagenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Repair and mutagenesis of herpes simplex virus in UV-irradiated monkey cells

    International Nuclear Information System (INIS)

    Lytle, C.D.; Goddard, J.G.; Lin, C.H.

    1980-01-01

    Mutagenic repair in mammalian cells was investigated by determining the mutagenesis of UV-irradiated or unirradiated herpes simplex virus in UV-irradiated CV-1 monkey kidney cells. These results were compared with the results for UV-enhanced virus reactivation (UVER) in the same experimental situation. High and low multiplicities of infection were used to determine the effects of multiplicity reactivation (MR). UVER and MR were readily demonstrable and were approximately equal in amount in an infectious center assay. For this study, a forward-mutation assay was developed to detect virus mutants resistant to iododeoxycytidine (ICdR), probably an indication of the mutant virus being defective at its thymidine kinase locus. ICdR-resistant mutants did not have a growth advantage over wild-type virus in irradiated or unirradiated cells. Thus, higher fractions of mutant virus indicated greater mutagenesis during virus repair and/or replication. The data showed that: (1) unirradiated virus was mutated in unirradiated cells, providing a background level of mutagenesis; (2) unirradiated virus was mutated about 40% more in irradiated cells, indicating that virus replication (DNA synthesis) became more mutagenic as a result of cell irradiation; (3) irradiated virus was mutated much more (about 6-fold) than unirradiated virus, even in unirradiated cells; (4) cell irradiation did not change the mutagenesis of irradiated virus except at high multiplicity of infection. High multiplicity of infection did not demonstrate UVER or MR alone to be either error-free or error-prone. When the two processes were present simultaneously, they were mutagenic. (orig.)

  4. Development of a high-frequency in vivo transposon mutagenesis system for Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942.

    Science.gov (United States)

    Watabe, Kazuyuki; Mimuro, Mamoru; Tsuchiya, Tohru

    2014-11-01

    Synechocystis sp. PCC 6803 (Synechocystis) is the first sequenced photosynthetic organism and has two advantages: natural transformation and light-activated heterotrophic growth. Such characteristics have mainly promoted reverse genetic analysis in this organism, however, to date approximately 50% of genes are still annotated as 'unknown protein' or 'hypothetical protein'. Therefore, forward genetic analysis is required for the identification of significant genes responsible for photosynthesis and other physiological phenomena among the genes of unknown function. The in vivo transposon mutagenesis system is one of the major methods for random mutagenesis. However, present in vivo transposon mutagenesis systems for cyanobacteria face problems such as relatively low frequency of transposition and repeated transposition in the host cells. In this study, we constructed vectors based on a mini-Tn5-derived vector that was designed to prevent repeated transposition. Our vectors carry a hyperactive transposase and optimized recognition sequence of transposase, which were reported to enhance frequency of transposition. Using the vector, we succeeded in highly frequent transposition (9×10(-3) per recipient cell) in Synechocystis. Transposon insertion sites of 10 randomly selected mutants indicated that the insertion sites spread throughout the genome with low sequence dependency. Furthermore, one of the 10 mutants exhibited the slow-growing phenotype, and the mutant was functionally complemented by using our expression vector. Our system also worked with another model cyanobacterium, Synechococcus elongatus PCC 7942, with high frequency. These results indicate that the developed system can be applied to the forward genetic analysis of a broad range of cyanobacteria. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  5. Transgenic Chinese hamster V79 cell lines which exhibit variable levels of gpt mutagenesis

    International Nuclear Information System (INIS)

    Klein, C.B.; Rossman, T.G.

    1990-01-01

    The Escherichia coli gpt gene coding for xanthine-guanine phosphoribosyl transferase has been stably transfected into HPRT - Chinese hamster V79 cells. Several gpt - cell lines have been established, which retain the sequence(s) even after long-term culture without selection for gpt. While spontaneous mutagenesis to gpt - occurs rather frequently for most cell lines, it cannot be correlated with either the number of plasmid integration sites or deletion of the plasmid sequence(s). One transgenic cell line (g12), which continuously maintains a low spontaneous mutation frequency was used in comparative mutagenesis studies with wild-type V79 cells (gpt vs. hprt). Alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and β-propiolactone (BPL) are shown to be equally toxic and mutagenic in both g12 and V79 cells. UV and X-rays are also equally toxic to both cell lines. The data presented here suggests that g12 cells may be useful to study mammalian mutagenesis by agents which yield limited response at the hprt locus

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

    Science.gov (United States)

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

    2014-04-01

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

  7. Spontaneous mutability and light-induced mutagenesis in Salmonella typhimurium: effects of an R-plasmid

    International Nuclear Information System (INIS)

    Valdivia, L.

    1979-01-01

    The UV-protecting plasmid R46 was transferred by conjugation to a genetically marked mouse-virulent Salmonella typhimurium strain, not derived from LT2; in this host the plasmid conferred UV protection and enhanced UV mutagenesis just as it does in LT2 lines. Tra - derivatives of R46 encountered during transduction retained UV-protecting and mutagenesis-enhancing ability. Stored strains carrying the R46-derived plasmids with strong mutator effect but not UV-protecting had lost most of their original streptomycin resistance but were slightly resistant to spectinomycin; attempts to transfer such plasmids failed. R46 enhanced the weak mutagenic effect of visible light on several his and trp mutants of strain LT2, including some whose frequency of spontaneous reversion was not increased by the plasmid. A mutagenic effect was produced by visible-light irradiation of hisG46(R46), either growing cells or nonmultiplying (histidine-deprived cells at 10 0 C). Presence of catalase or cyanide during irradiation did not prevent mutagenesis, which excludes some hypothetical mechanisms. Visible-light irradiation of hisG46 or hisG46(R46) under strict anaerobiosis had little or no mutagenic effect (controls showed that revertants if produced would have been detected). This is as expected if visible-light irradiation in air causes photodynamic damage to DNA and mutations are produced during error-prone, plasmid-enhanced repair

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

    Science.gov (United States)

    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. © 2016 Institute of Zoology, Chinese Academy of Sciences.

  9. Insertional Mutagenesis by a Hybrid PiggyBac and Sleeping Beauty Transposon in the Rat

    Science.gov (United States)

    Furushima, Kenryo; Jang, Chuan-Wei; Chen, Diane W.; Xiao, Ningna; Overbeek, Paul A.; Behringer, Richard R.

    2012-01-01

    A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat. PMID:23023007

  10. Combined mutagenesis of Rhodosporidium toruloides for improved production of carotenoids and lipids.

    Science.gov (United States)

    Zhang, Chaolei; Shen, Hongwei; Zhang, Xibin; Yu, Xue; Wang, Han; Xiao, Shan; Wang, Jihui; Zhao, Zongbao K

    2016-10-01

    To improve production of lipids and carotenoids by the oleaginous yeast Rhodosporidium toruloides by screening mutant strains. Upon physical mutagenesis of the haploid strain R. toruloides np11 with an atmospheric and room temperature plasma method followed by chemical mutagenesis with nitrosoguanidine, a mutant strain, R. toruloides XR-2, formed dark-red colonies on a screening plate. When cultivated in nitrogen-limited media, XR-2 cells grew slower but accumulated 0.23 g lipids/g cell dry wt and 0.75 mg carotenoids/g CDW. To improve its production capacity, different amino acids and vitamins were supplemented. p-Aminobenzoic acid and tryptophan had beneficial effects on cell growth. When cultivated in nitrogen-limited media in the presence of selected vitamins, XR-2 accumulated 0.41 g lipids/g CDW and 0.69 mg carotenoids/g CDW. A mutant R. toruloides strain with improved production profiles for lipids and carotenoids was obtained, indicating its potential to use combined mutagenesis for a more productive phenotype.

  11. Polyamines modulate carcinogen-induced mutagenesis in vivo.

    Science.gov (United States)

    Wallon, U Margaretha; O'Brien, Thomas G

    2005-01-01

    Elevated polyamine levels as a consequence of targeted overexpression of ornithine decarboxylase (ODC) to murine skin enhance susceptibility to tumorigenesis in this tissue. A possible mechanism for the enhanced susceptibility phenotype is an increased sensitivity of tissues with elevated polyamine levels to the mutagenic action of carcinogens. To test this hypothesis, a transgenic mouse model containing the Big Blue transgene and also expressing a K6/ODC transgene was developed. Incorporation of the K6/ODC transgene into the Big Blue model did not affect the spontaneous lacI mutant frequency in either skin or epidermis of the double-transgenic mice. After skin treatment with single doses of either 7,12-dimethylbenz[a]anthracene or N-methyl-N'-nitro-N-nitrosoguanidine, however, the mutant frequency was significantly increased in the skin of double-transgenic Big Blue;K6/ODC mice compared to Big Blue controls. The increases in mutant frequency were clearly due to ODC transgene activity, since treatment of mice with the ODC inhibitor, alpha-difluoromethylornithine, completely abolished the difference in mutant frequencies between double-transgenic and Big Blue mice. These results demonstrate that intracellular polyamine levels modulate mutation induction following carcinogen exposure. 2004 Wiley-Liss, Inc.

  12. Accurate prediction of stability changes in protein mutants by combining machine learning with structure based computational mutagenesis.

    Science.gov (United States)

    Masso, Majid; Vaisman, Iosif I

    2008-09-15

    Accurate predictive models for the impact of single amino acid substitutions on protein stability provide insight into protein structure and function. Such models are also valuable for the design and engineering of new proteins. Previously described methods have utilized properties of protein sequence or structure to predict the free energy change of mutants due to thermal (DeltaDeltaG) and denaturant (DeltaDeltaG(H2O)) denaturations, as well as mutant thermal stability (DeltaT(m)), through the application of either computational energy-based approaches or machine learning techniques. However, accuracy associated with applying these methods separately is frequently far from optimal. We detail a computational mutagenesis technique based on a four-body, knowledge-based, statistical contact potential. For any mutation due to a single amino acid replacement in a protein, the method provides an empirical normalized measure of the ensuing environmental perturbation occurring at every residue position. A feature vector is generated for the mutant by considering perturbations at the mutated position and it's ordered six nearest neighbors in the 3-dimensional (3D) protein structure. These predictors of stability change are evaluated by applying machine learning tools to large training sets of mutants derived from diverse proteins that have been experimentally studied and described. Predictive models based on our combined approach are either comparable to, or in many cases significantly outperform, previously published results. A web server with supporting documentation is available at http://proteins.gmu.edu/automute.

  13. Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

    Energy Technology Data Exchange (ETDEWEB)

    Cook, Melloni N. [University of Memphis; Dunning, Jonathan P [University of Memphis; Wiley, Ronald G [Vanderbilt University and Veterans Administration, Nashville, TN; Chesler, Elissa J [ORNL; Johnson, Dabney K [ORNL; Goldowitz, Daniel [University of Tennessee Health Science Center, Memphis

    2007-01-01

    We report on a behavioral screening test battery that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and employed a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open field mutants (one displaying hyper-locomotion, the other hypo-locomotion), four tail suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsiveness to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning and memory mutant (displaying reduced response to the conditioned stimulus) These findings highlight the utility of a set of behavioral tasks used in a high throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.

  14. Model of a DNA-protein complex of the architectural monomeric protein MC1 from Euryarchaea.

    Directory of Open Access Journals (Sweden)

    Françoise Paquet

    Full Text Available In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1 from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.

  15. High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP receptor expression and function.

    Directory of Open Access Journals (Sweden)

    Anke Bill

    Full Text Available The human prostacyclin receptor (hIP receptor is a seven-transmembrane G protein-coupled receptor (GPCR that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

  16. Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress.

    Science.gov (United States)

    LeBlanc, Chantal; Zhang, Fei; Mendez, Josefina; Lozano, Yamile; Chatpar, Krishna; Irish, Vivian F; Jacob, Yannick

    2018-01-01

    The CRISPR/Cas9 system has greatly improved our ability to engineer targeted mutations in eukaryotic genomes. While CRISPR/Cas9 appears to work universally, the efficiency of targeted mutagenesis and the adverse generation of off-target mutations vary greatly between different organisms. In this study, we report that Arabidopsis plants subjected to heat stress at 37°C show much higher frequencies of CRISPR-induced mutations compared to plants grown continuously at the standard temperature (22°C). Using quantitative assays relying on green fluorescent protein (GFP) reporter genes, we found that targeted mutagenesis by CRISPR/Cas9 in Arabidopsis is increased by approximately 5-fold in somatic tissues and up to 100-fold in the germline upon heat treatment. This effect of temperature on the mutation rate is not limited to Arabidopsis, as we observed a similar increase in targeted mutations by CRISPR/Cas9 in Citrus plants exposed to heat stress at 37°C. In vitro assays demonstrate that Cas9 from Streptococcus pyogenes (SpCas9) is more active in creating double-stranded DNA breaks at 37°C than at 22°C, thus indicating a potential contributing mechanism for the in vivo effect of temperature on CRISPR/Cas9. This study reveals the importance of temperature in modulating SpCas9 activity in eukaryotes, and provides a simple method to increase on-target mutagenesis in plants using CRISPR/Cas9. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  17. Retroviral insertional mutagenesis identifies genes that collaborate with NUP98-HOXD13 during leukemic transformation.

    Science.gov (United States)

    Slape, Christopher; Hartung, Helge; Lin, Ying-Wei; Bies, Juraj; Wolff, Linda; Aplan, Peter D

    2007-06-01

    The t(2;11)(q31;p15) chromosomal translocation results in a fusion between the NUP98 and HOXD13 genes and has been observed in patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia. We previously showed that expression of the NUP98-HOXD13 (NHD13) fusion gene in transgenic mice results in an invariably fatal MDS; approximately one third of mice die due to complications of severe pancytopenia, and about two thirds progress to a fatal acute leukemia. In the present study, we used retroviral insertional mutagenesis to identify genes that might collaborate with NHD13 as the MDS transformed to an acute leukemia. Newborn NHD13 transgenic mice and littermate controls were infected with the MOL4070LTR retrovirus. The onset of leukemia was accelerated, suggesting a synergistic effect between the NHD13 transgene and the genes neighboring retroviral insertion events. We identified numerous common insertion sites located near protein-coding genes and confirmed dysregulation of a subset of these by expression analyses. Among these genes were Meis1, a known collaborator of HOX and NUP98-HOX fusion genes, and Mn1, a transcriptional coactivator involved in human leukemia through fusion with the TEL gene. Other putative collaborators included Gata2, Erg, and Epor. Of note, we identified a common insertion site that was >100 kb from the nearest coding gene, but within 20 kb of the miR29a/miR29b1 microRNA locus. Both of these miRNA were up-regulated, demonstrating that retroviral insertional mutagenesis can target miRNA loci as well as protein-coding loci. Our data provide new insights into NHD13-mediated leukemogenesis as well as retroviral insertional mutagenesis mechanisms.

  18. The Structure of Urease Activation Complexes Examined by Flexibility Analysis, Mutagenesis, and Small-angle X-ray Scattering Approaches

    International Nuclear Information System (INIS)

    Quiroz, Soledad; Sukuru, Sai Chetan K.; Hausinger, Robert P.; Kuhn, Leslie A.; Heller, William T

    2008-01-01

    Conformational changes of Klebsiella aerogenes urease apoprotein (UreABC) 3 induced upon binding of the UreD and UreF accessory proteins were examined by a combination of flexibility analysis, mutagenesis, and small-angle X-ray scattering (SAXS). ProFlex analysis of urease provided evidence that the major domain of UreB can move in a hinge-like motion to account for prior chemical cross-linking results. Rigidification of the UreB hinge region, accomplished through a G11P mutation, reduced the extent of urease activation, in part by decreasing the nickel content of the mutant enzyme, and by sequestering a portion of the urease apoprotein in a novel activation complex that includes all of the accessory proteins. SAXS analyses of urease, (UreABC-UreD) 3 , and (UreABC-UreDF) 3 confirm that UreD and UreF bind near UreB at the periphery of the (UreAC) 3 structure. This study supports an activation model in which a domain-shifted UreB conformation in (UreABC-UreDF) 3 allows CO 2 and nickel ions to gain access to the nascent active site

  19. Sulforaphane induces phase II detoxication enzymes in mouse skin and prevents mutagenesis induced by a mustard gas analog

    Energy Technology Data Exchange (ETDEWEB)

    Abel, E.L. [Department of Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Science Park, Smithville, TX 78957 (United States); Boulware, S. [Division of Pharmacy and Toxicology, College of Pharmacy, The University of Texas at Austin, Dell Pediatric Research Institute, 1400 Barbara Jordan Blvd., Austin, TX 78723 (United States); Fields, T.; McIvor, E.; Powell, K.L. [Department of Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Science Park, Smithville, TX 78957 (United States); DiGiovanni, J.; Vasquez, K.M. [Division of Pharmacy and Toxicology, College of Pharmacy, The University of Texas at Austin, Dell Pediatric Research Institute, 1400 Barbara Jordan Blvd., Austin, TX 78723 (United States); MacLeod, M.C., E-mail: mcmacleod@mdanderson.org [Department of Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Science Park, Smithville, TX 78957 (United States)

    2013-02-01

    Mustard gas, used in chemical warfare since 1917, is a mutagenic and carcinogenic agent that produces severe dermal lesions for which there are no effective therapeutics; it is currently seen as a potential terrorist threat to civilian populations. Sulforaphane, found in cruciferous vegetables, is known to induce enzymes that detoxify compounds such as the sulfur mustards that react through electrophilic intermediates. Here, we observe that a single topical treatment with sulforaphane induces mouse epidermal levels of the regulatory subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, and also increases epidermal levels of reduced glutathione. Furthermore, a glutathione S-transferase, GSTA4, is also induced in mouse skin by sulforaphane. In an in vivo model in which mice are given a single mutagenic application of the sulfur mustard analog 2-(chloroethyl) ethyl sulfide (CEES), we now show that therapeutic treatment with sulforaphane abolishes the CEES-induced increase in mutation frequency in the skin, measured four days after exposure. Sulforaphane, a natural product currently in clinical trials, shows promise as an effective therapeutic against mustard gas. -- Highlights: ► Sulforaphane induces increased levels of glutathione in mouse skin. ► Sulforaphane induces increased levels of GSTA4 in mouse skin. ► Sulforaphane, applied after CEES-treatment, completely abolishes CEES-mutagenesis. ► The therapeutic effect may suggest a long biological half-life for CEES in vivo.

  20. Sulforaphane induces phase II detoxication enzymes in mouse skin and prevents mutagenesis induced by a mustard gas analog

    International Nuclear Information System (INIS)

    Abel, E.L.; Boulware, S.; Fields, T.; McIvor, E.; Powell, K.L.; DiGiovanni, J.; Vasquez, K.M.; MacLeod, M.C.

    2013-01-01

    Mustard gas, used in chemical warfare since 1917, is a mutagenic and carcinogenic agent that produces severe dermal lesions for which there are no effective therapeutics; it is currently seen as a potential terrorist threat to civilian populations. Sulforaphane, found in cruciferous vegetables, is known to induce enzymes that detoxify compounds such as the sulfur mustards that react through electrophilic intermediates. Here, we observe that a single topical treatment with sulforaphane induces mouse epidermal levels of the regulatory subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis, and also increases epidermal levels of reduced glutathione. Furthermore, a glutathione S-transferase, GSTA4, is also induced in mouse skin by sulforaphane. In an in vivo model in which mice are given a single mutagenic application of the sulfur mustard analog 2-(chloroethyl) ethyl sulfide (CEES), we now show that therapeutic treatment with sulforaphane abolishes the CEES-induced increase in mutation frequency in the skin, measured four days after exposure. Sulforaphane, a natural product currently in clinical trials, shows promise as an effective therapeutic against mustard gas. -- Highlights: ► Sulforaphane induces increased levels of glutathione in mouse skin. ► Sulforaphane induces increased levels of GSTA4 in mouse skin. ► Sulforaphane, applied after CEES-treatment, completely abolishes CEES-mutagenesis. ► The therapeutic effect may suggest a long biological half-life for CEES in vivo.

  1. Pharmacological and functional characterisation of the wild-type and site-directed mutants of the human H1 histamine receptor stably expressed in CHO cells.

    Science.gov (United States)

    Moguilevsky, N; Varsalona, F; Guillaume, J P; Noyer, M; Gillard, M; Daliers, J; Henichart, J P; Bollen, A

    1995-01-01

    A cDNA clone for the human histamine H1 receptor was isolated from a lung cDNA library and stably expressed in CHO cells. The recombinant receptor protein present in the cell membranes, displayed the functional and binding characteristics of histamine H1 receptors. Mutation of Ser155 to Ala in the fourth transmembrane domain did not significantly change the affinity of the receptor for histamine and H1 antagonists. However, mutation of the fifth transmembrane Asn198 to Ala resulted in a dramatic decrease of the affinity for histamine binding, and for the histamine-induced polyphosphoinositides breakdown, whereas the affinity towards antagonists was not significantly modified. In addition, mutation of another fifth transmembrane amino acid, Thr194 to Ala also diminished, but to a lesser extent, the affinity for histamine. These data led us to propose a molecular model for histamine interaction with the human H1 receptor. In this model, the amide moiety of Asn198 and the hydroxyl group of Thr194 are involved in hydrogen bonding with the nitrogen atoms of the imidazole ring of histamine. Moreover, mutation of Thr194 to Ala demonstrated that this residue is responsible for the discrimination between enantiomers of cetirizine.

  2. Improvement of the activity and thermostability of microbial transglutaminase by multiple-site mutagenesis.

    Science.gov (United States)

    Mu, Dongdong; Lu, Jiaojiao; Shu, Chang; Li, Haowen; Li, Xingjiang; Cai, Jing; Luo, Shuizhong; Yang, Peizhou; Jiang, Shaotong; Zheng, Zhi

    2018-01-01

    Microbial transglutaminase (MTG) is an enzyme widely used in the food industry. Mutiple-site mutagenesis of Streptomyces mobaraensis transglutaminase was performed in Escherichia coli. According to enzymatic assay and thermostability study, among three penta-site MTG mutants (DM01-03), DM01 exhibited the highest enzymatic activity of 55.7 ± 1.4 U/mg and longest half-life at 50 °C (418.2 min) and 60 °C (24.8 min).

  3. Uvm mutants of Escherichia coli K 12 deficient in UV mutagenesis. Pt. 1

    International Nuclear Information System (INIS)

    Steinborn, G.

    1978-01-01

    Selection for defective reversion induction, after UV treatment of E. coli K 12, yielded uvm mutants. These mutants exhibited highly reduced or no UV mutability for all loci tested although they were moderately and normally mutable by X-rays and EMS, respectively. Uvm mutations confer only a slight sensitivity to killing by UV and X-rays and no clear sensitivity to the lethal effect of HN2, EMS or MMS. Growth and viability of untreated uvm cells were normal. The properties of uvm mutants are discussed in relation to those of other relevant mutant types and to some actual problems of induced mutagenesis. (orig.) 891 AJ [de

  4. Use of CRISPR/Cas Genome Editing Technology for Targeted Mutagenesis in Rice.

    Science.gov (United States)

    Xu, Rongfang; Wei, Pengcheng; Yang, Jianbo

    2017-01-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system is a newly emerging mutagenesis (gene-editing) tool in genetic engineering. Among the agriculturally important crops, several genes have been successfully mutated by the system, and some agronomic important traits have been rapidly generated, which indicates the potential applications in both scientific research and plant breeding. In this chapter, we describe a standard gene-editing procedure to effectively target rice genes and to make specific rice mutants using the CRISPR/Cas9 system mediated by Agrobacterium transformation.

  5. Modelling of three-dimensional structures of cytochromes P450 11B1 and 11B2.

    Science.gov (United States)

    Belkina, N V; Lisurek, M; Ivanov, A S; Bernhardt, R

    2001-12-15

    The final steps of the biosynthesis of glucocorticoids and mineralocorticoids in the adrenal cortex require the action of two different cytochromes P450--CYP11B1 and CYP11B2. Homology modelling of the three-dimensional structures of these cytochromes was performed based on crystallographic coordinates of two bacterial P450s, CYP102 (P450BM-3) and CYP108 (P450terp). Principal attention was given to the modelling of the active sites and a comparison of the active site structures of CYP11B1 and CYP11B2 was performed. It can be demonstrated that key residue contacts within the active site appear to depend on the orientation of the heme. The obtained 3D structures of CYP11B1 and CYP11B2 were used for investigation of structure-function relationships of these enzymes. Previously obtained results on naturally occurring mutants and on mutants obtained by site-directed mutagenesis are discussed.

  6. A homology-based model of the human 5-HT2A receptor derived from an in silico activated G-protein coupled receptor

    Science.gov (United States)

    Chambers, James J.; Nichols, David E.

    2002-07-01

    A homology-based model of the 5-HT2A receptor was produced utilizing an activated form of the bovine rhodopsin (Rh) crystal structure [1,2]. In silico activation of the Rh structure was accomplished by isomerization of the 11- cis-retinal (1) chromophore, followed by constrained molecular dynamics to relax the resultant high energy structure. The activated form of Rh was then used as a structural template for development of a human 5-HT2A receptor model. Both the 5-HT2A receptor and Rh are members of the G-protein coupled receptor (GPCR) super-family. The resulting homology model of the receptor was then used for docking studies of compounds representing a cross-section of structural classes that activate the 5-HT2A receptor, including ergolines, tryptamines, and amphetamines. The ligand/receptor complexes that ensued were refined and the final binding orientations were observed to be compatible with much of the data acquired through both diversified ligand design and site directed mutagenesis.

  7. HIV-1 entry inhibition by small-molecule CCR5 antagonists: A combined molecular modeling and mutant study using a high-throughput assay

    International Nuclear Information System (INIS)

    Labrecque, Jean; Metz, Markus; Lau, Gloria; Darkes, Marilyn C.; Wong, Rebecca S.Y.; Bogucki, David; Carpenter, Bryon; Chen Gang; Li Tongshuang; Nan, Susan; Schols, Dominique; Bridger, Gary J.; Fricker, Simon P.; Skerlj, Renato T.

    2011-01-01

    Based on the attrition rate of CCR5 small molecule antagonists in the clinic the discovery and development of next generation antagonists with an improved pharmacology and safety profile is necessary. Herein, we describe a combined molecular modeling, CCR5-mediated cell fusion, and receptor site-directed mutagenesis approach to study the molecular interactions of six structurally diverse compounds (aplaviroc, maraviroc, vicriviroc, TAK-779, SCH-C and a benzyloxycarbonyl-aminopiperidin-1-yl-butane derivative) with CCR5, a coreceptor for CCR5-tropic HIV-1 strains. This is the first study using an antifusogenic assay, a model of the interaction of the gp120 envelope protein with CCR5. This assay avoids the use of radioactivity and HIV infection assays, and can be used in a high throughput mode. The assay was validated by comparison with other established CCR5 assays. Given the hydrophobic nature of the binding pocket several binding models are suggested which could prove useful in the rational drug design of new lead compounds.

  8. Molecularly imprinted protein recognition cavities bearing exchangeable binding sites for postimprinting site-directed introduction of reporter molecules for readout of binding events.

    Science.gov (United States)

    Sunayama, Hirobumi; Takeuchi, Toshifumi

    2014-11-26

    Protein-imprinted cavities bearing exchangeable domains to be used for postimprinting fluorophore introduction to transform binding events into fluorescence changes were constructed in molecularly imprinted polymer (MIPs) matrixes prepared on glass substrates. Copolymerization was performed with acrylamide, N,N'-methylenebisaclylamide, and a newly designed functional group-exchangeable monomer, ({[2-(2-methacrylamido)ethyldithio]ethylcarbamoyl}methoxy)acetic acid (MDTA), in the presence of a model basic protein, lysozyme (Lyso); MDTA can interact with Lyso and assemble close to Lyso in the resulting polymer. After removal of Lyso, followed by a disulfide reduction to cleave the (ethylcarbamoylmethoxy)acetic acid moiety from the MDTA residues, the exposed thiol groups within the imprinted cavities were modified by aminoethylpyridyldisulfide to be transformed into aminoethyl groups that function as active sites for amine-reactive fluorophores. Fluorescein isothiocyanate (FITC) was then coupled with the aminoethyl groups, yielding site specifically FITC-modified signaling imprinted cavities for Lyso binding. Because the in-cavity fluorescent labeling was achieved via a disulfide linkage, it was easy to remove, exchange, and/or replace amine-reactive fluorophores. This facilitated the screening of fluorophores to select the highest readout for binding events, replace fluorophores when photobleaching occurred, and introduce other functions. The proposed molecular imprinting process, combined with postimprinting modifications, is expected to provide an affordable route to develop multifunctional MIPs for specific detection of protein binding events.

  9. Subunit organization in the TatA complex of the twin arginine protein translocase: a site-directed EPR spin labeling study.

    Science.gov (United States)

    White, Gaye F; Schermann, Sonya M; Bradley, Justin; Roberts, Andrew; Greene, Nicholas P; Berks, Ben C; Thomson, Andrew J

    2010-01-22

    The Tat system is used to transport folded proteins across the cytoplasmic membrane in bacteria and archaea and across the thylakoid membrane of plant chloroplasts. Multimers of the integral membrane TatA protein are thought to form the protein-conducting element of the Tat pathway. Nitroxide radicals were introduced at selected positions within the transmembrane helix of Escherichia coli TatA and used to probe the structure of detergent-solubilized TatA complexes by EPR spectroscopy. A comparison of spin label mobilities allowed classification of individual residues as buried within the TatA complex or exposed at the surface and suggested that residues Ile(12) and Val(14) are involved in interactions between helices. Analysis of inter-spin distances suggested that the transmembrane helices of TatA subunits are arranged as a single-walled ring containing a contact interface between Ile(12) on one subunit and Val(14) on an adjacent subunit. Experiments in which labeled and unlabeled TatA samples were mixed demonstrate that TatA subunits are exchanged between TatA complexes. This observation is consistent with the TatA dynamic polymerization model for the mechanism of Tat transport.

  10. Topology of AspT, the Aspartate:Alanine Antiporter of Tetragenococcus halophilus, Determined by Site-Directed Fluorescence Labeling▿ †

    Science.gov (United States)

    Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C.; Abe, Keietsu

    2007-01-01

    The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of l-aspartate (Asp) with release of l-alanine (Ala) and CO2. The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an l-aspartate-β-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity. PMID:17660287

  11. Evidence for a Rad18-independent frameshift mutagenesis pathway in human cell-free extracts.

    Directory of Open Access Journals (Sweden)

    Régine Janel-Bintz

    Full Text Available Bypass of replication blocks by specialized DNA polymerases is crucial for cell survival but may promote mutagenesis and genome instability. To gain insight into mutagenic sub-pathways that coexist in mammalian cells, we examined N-2-acetylaminofluorene (AAF-induced frameshift mutagenesis by means of SV40-based shuttle vectors containing a single adduct. We found that in mammalian cells, as previously observed in E. coli, modification of the third guanine of two target sequences, 5'-GGG-3' (3G and 5'-GGCGCC-3' (NarI site, induces -1 and -2 frameshift mutations, respectively. Using an in vitro assay for translesion synthesis, we investigated the biochemical control of these events. We showed that Pol eta, but neither Pol iota nor Pol zeta, plays a major role in the frameshift bypass of the AAF adduct located in the 3G sequence. By complementing PCNA-depleted extracts with either a wild-type or a non-ubiquitinatable form of PCNA, we found that this Pol eta-mediated pathway requires Rad18 and ubiquitination of PCNA. In contrast, when the AAF adduct is located within the NarI site, TLS is only partially dependent upon Pol eta and Rad18, unravelling the existence of alternative pathways that concurrently bypass this lesion.

  12. In vitro mutagenesis and identification of mutants via ISSR in lily (Lilium longiflorum).

    Science.gov (United States)

    Xi, Mengli; Sun, Lina; Qiu, Shuai; Liu, Juanjuan; Xu, Jin; Shi, Jisen

    2012-06-01

    An efficient in vitro mutagenesis protocol for Lilium longiflorum Thunb. cv. White fox has been established. The effect of 6-BA and NAA on adventitious bud formation from the bulblet-scale thin cell layers was tested. Results showed that the optimal medium for adventitious bud induction is MS basal medium supplemented with 2.0 mg/l 6-BA and 0.1 mg/l NAA. The differentiation frequency and the average number of adventitious buds reached 95.55% and 3.00, respectively. Various doses (0.0, 0.5, 1.0, 1.5, 2.0, and 2.5 Gy) of gamma rays were applied to investigate the effect of radiation on adventitious bud formation from bulblet-scale thin cell layers. The forming capacity of the adventitious buds significantly decreased with the increase of radiation dose. The results suggested that the optimal irradiation dose is 1.0 Gy. Dose of 1.0 Gy treatment resulted in 55.33% survival of irradiated bulblet-scale thin cell layers and 39.27% mutagenesis rate. The genetic variations among the morphological mutants were evaluated by DNA fingerprinting using ISSR molecular marker. The genetic variation frequency reached 36.06% using seven ISSR primers. Out of the 50 mutant lines transferred to the greenhouse, 9 were observed to have significantly different morphological characters than those of the controls.

  13. A mutagenesis-derived broad-spectrum disease resistance locus in wheat.

    Science.gov (United States)

    Campbell, Jackie; Zhang, Hongtao; Giroux, Michael J; Feiz, Leila; Jin, Yue; Wang, Meinan; Chen, Xianming; Huang, Li

    2012-07-01

    Wheat leaf rust, stem rust, stripe rust, and powdery mildew caused by the fungal pathogens Puccinia triticina, P. graminis f. sp. tritici, P. striiformis f. sp. tritici, and Blumeria graminis f. sp. tritici, respectively, are destructive diseases of wheat worldwide. Breeding durable disease resistance cultivars rely largely on continually introgressing new resistance genes, especially the genes with different defense mechanisms, into adapted varieties. Here, we describe a new resistance gene obtained by mutagenesis. The mutant, MNR220 (mutagenesis-derived new resistance), enhances resistance to three rusts and powdery mildew, with the characteristics of delayed disease development at the seedling stage and completed resistance at the adult plant stage. Genetic analysis demonstrated that the resistance in MNR220 is conferred by a single semidominant gene mapped on the short arm of chromosome 2B. Gene expression profiling of several pathogenesis-related genes indicated that MNR220 has an elevated and rapid pathogen-induced response. In addition to its potential use in breeding for resistance to multiple diseases, high-resolution mapping and cloning of the disease resistance locus in MNR220 may lead to a better understanding of the regulation of defense responses in wheat.

  14. Characterization of a metagenome-derived protease from contaminated agricultural soil microorganisms and its random mutagenesis.

    Science.gov (United States)

    Jiang, Chengjian; Zhang, Liang; Li, Fajia; Meng, Can; Zeng, Rong; Deng, Jie; Shen, Peihong; Ou, Qian; Wu, Bo

    2017-11-01

    Proteases are typical key enzymes that hydrolyze proteins into amino acids and peptides. Numerous proteases have been studied, but the discovery of metagenome-derived proteases is still significant for both commercial applications and basic research. An unexplored protease gene sep1A was identified by function-based screening from a plasmid metagenomic library derived from uncultured contaminated agricultural soil microorganisms. The putative protease gene was subcloned into pET-32a (+) vector and overexpressed in E. coli BL21(DE3) pLysS, then the recombinant protein was purified to homogeneity. The detailed biochemical characterization of the Sep1A protein was performed, including its molecular characterization, specific activity, pH-activity profile, metal ion-activity profile, and enzyme kinetic assays. Furthermore, the protein engineering approach of random mutagenesis via error-prone PCR was applied on the original Sep1A protein. Biochemical characterization demonstrated that the purified recombinant Ep48 protein could hydrolyze casein. Compared with the original Sep1A protein, the best variant of Ep48 in the random mutagenesis library, with the Gln307Leu and Asp391Gly changes, exhibited 2.62-fold activity at the optimal reaction conditions of 50 °C and pH 9.0. These results are the first step toward a better understanding of the properties of Sep1A protein. Protein engineering with error-prone PCR paves the way toward the metagenome-derived genes for biotechnological applications.

  15. The effect of chronic alcohol consumption on mitochondrial DNA mutagenesis in human blood

    Energy Technology Data Exchange (ETDEWEB)

    Wurmb-Schwark, N. von [Institute of Legal Medicine, Christian Albrecht University of Kiel, Arnold-Heller-Str. 12, 24105 Kiel (Germany)], E-mail: nvonwurmb@rechtsmedizin.uni-kiel.de; Ringleb, A.; Schwark, T. [Institute of Legal Medicine, Christian Albrecht University of Kiel, Arnold-Heller-Str. 12, 24105 Kiel (Germany); Broese, T.; Weirich, S.; Schlaefke, D. [Clinic of Psychiatry and Psychotherapy, University of Rostock, Gehlsheimer Str. 20, Rostock (Germany); Wegener, R. [Institute of Legal Medicine, St-Georg-Str. 108, University of Rostock, 18055 Rostock (Germany); Oehmichen, M. [Institute of Legal Medicine, Christian Albrecht University of Kiel, Arnold-Heller-Str. 12, 24105 Kiel (Germany)

    2008-01-01

    The 4977 bp deletion of mitochondrial DNA (mtDNA) is known to accumulate with increasing age in post mitotic tissues. Recently, studies came out detecting this specific alteration also in fast replicating cells, e.g. in blood or skin tissue, often in correlation to specific diseases or - specifically in skin - external stressors such as UV radiation. In this study, we investigated mitochondrial mutagenesis in 69 patients with a chronic alcoholic disease and 46 age matched controls with a moderate drinking behavior. Two different fragments, specific for total and for deleted mtDNA (dmtDNA) were amplified in a duplex-PCR. A subsequent fragment analysis was performed and for relative quantification, the quotient of the peak areas of amplification products specific for deleted and total mtDNA was determined. Additionally, a real time PCR was performed to quantify mtDNA copy number. The relative amount of 4977 bp deleted mtDNA in alcoholics was significantly increased compared to controls. On the other hand, no difference regarding the mtDNA/nuclear DNA ratio in both investigated groups was detected. Additionally, no age dependence could be found nor in alcoholics, neither in the control group. These findings indicate that mtDNA mutagenesis in blood can be influenced by stressors such as alcohol. Ethanol seems to be a significant factor to alter mitochondrial DNA in blood and might be an additional contributor for the cellular aging process.

  16. Directed mutagenesis under the action of mobile elements in unstable lines of Drosophila melanogaster

    International Nuclear Information System (INIS)

    Gerasimova, T.I.; Mizrokhi, L.Yu.; Obolenkova, L.A.; Georgiev, P.G.

    1985-01-01

    The unstable mutation ct/sup MR2/, obtained under hybrid dysgenesis and caused by the incorporation of MDG 4 into locus cut, was described earlier. A peculiarity of this line and its derivatives is that multiple transpositions of different mobile elements, the transposition spurts, take place in them with a frequency of 10 -2 -10 -4 . A consequence of this process is active insertion mutagenesis taking place under the action of different transposons with high locus specifity. The mutations are unstable and frequently revert to wild type. However, there are some unstable revertants in which the mobile element is eliminated almost completely, and yet unstable mutations appear at the same locus at a high rate. In the case of cut locus, this happens due to repeated integration of MDG 4. The authors report cases of such repeated directed mutagenesis for several loci. Reversions were observed in the homozygous line as well as during the process of obtaining homozygous lines of new mutations using the chromosome with multiple inversions FM4, Y/sup 31d/sc 8 dmB. In situ hybridization with the [ 3 H]-labeled DNA was carried out as described earlier. The plasmid DNA containing cloned MDG4 and their long terminal repeats were used as probes for hybridization. Hybridization was done on the salivary gland polytene chromosomes of larvae. The new spontaneous mutations appearing in the process of hybridization were tested for allelism with the standard mutations y, w, cm, ct, D, and g

  17. Mutagenesis by damaged deoxyribonucleotides and its prevention by MutT-type hydrolyzing enzymes.

    Science.gov (United States)

    Kamiya, Hiroyuki; Satou, Kazuya; Hori, Mika; Iida, Emiko; Ishiguro, Chieko; Harashima, Hideyoshi

    2004-01-01

    Oxidized deoxyribonucleotides, 2-hydroxydeoxyadenosine 5'-triphosphate (2-OH-dATP) and 8-hydroxydeoxyguanosine 5'-triphosphate (8-OH-dGTP), were introduced into Escherichia coli strains deficient in DNA polymerase IV (a Y-family DNA polymerase encoded in the dinB gene), and the MutT and Orfl35 proteins to examine their in vivo roles in mutagenesis elicited by 2-OH-dATP and 8-OH-dGTP. 2-OH-dATP elicited mutations less efficiently in the dinB- strain than in the wild type strain, suggesting involvement of DNA polymerase IV in 2-OH-dATP-induced mutations. 8-OH-dGTP and 2-OH-dATP elicited mutations more efficiently in mutT- and orfl35- strains, respectively, than those in their isogenic mutT+ and orfl35+ strains. These results indicate that these proteins play important roles in mutagenesis induced by 2-OH-dATP and 8-OH-dGTP in vivo.

  18. Random mutagenesis of aspergillus niger and process optimization for enhanced production of glucose oxidase

    International Nuclear Information System (INIS)

    Haq, I.; Nawaz, A.; Mukhtar, A.N.H.; Mansoor, H.M.Z.; Ameer, S.M.

    2014-01-01

    The study deals with the improvement of wild strain Aspergillus niger IIB-31 through random mutagenesis using chemical mutagens. The main aim of the work was to enhance the glucose oxidase (GOX) yield of wild strain (24.57+-0.01 U/g of cell mass) through random mutagenesis and process optimization. The wild strain of Aspergillus niger IIB-31 was treated with chemical mutagens such as Ethyl methane sulphonate (EMS) and nitrous acid for this purpose. Mutagen treated 98 variants indicating the positive results were picked and screened for the glucose oxidase production using submerged fermentation. EMS treated E45 mutant strain gave the highest glucose oxidase production (69.47 + 0.01 U/g of cell mass), which was approximately 3-folds greater than the wild strain IIB-31. The preliminary cultural conditions for the production of glucose oxidase using submerged fermentation from strain E45 were also optimized. The highest yield of GOD was obtained using 8% glucose as carbon and 0.3% peptone as nitrogen source at a medium pH of 7.0 after an incubation period of 72 hrs at 30 degree. (author)

  19. Novel patterns of ultraviolet mutagenesis and Weigle reactivation in Staphylococcus aureus and phage phi II

    International Nuclear Information System (INIS)

    Thompson, J.K.; Hart, M.G.R.

    1981-01-01

    The effects of u.v. irradiation on the survival of Staphylococcus aureus and its phage phi11 were studied. The recA and uvr mutations affected their survival like synonymous mutations in Escherichia coli. Weigle reactivation (W-reactivation) of phi11 occurred in wild-type S. aureus and in a uvr mutant. Reactivation was recA-dependent and was accompanied by u.v.-induced mutagenesis in a temperature-sensitive mutant of phi11. Bacterial mutation to streptomycin resistance was induced by u.v. and was also recA-dependent. In S. aureus, as in E. coli, u.v. was a more effective mutagen in the uvr genetic background. However, a dose-squared response for u.v.-induced mutation of wild-type and uvr strains of S. aureus to streptomycin resistance, and of a trp auxotroph to tryptophan independence, was found only with u.v. doses below 1 J m -2 . In relation to the Uvr mechanism of DNA repair, u.v. mutagenesis in S. aureus may involve both repairable and non-repairable lesions. As in E. Coli, the uvr genetic background reduced the u.v. dose required for maximal W-reactivation of u.v.-irradiated phage. However, there was no enhancement of W-reactivation by post-irradiation broth incubation of S. aureus. The results are compatible with a non-inducible mechanism for this phenomenon. (author)

  20. Structural models of human eEF1A1 and eEF1A2 reveal two distinct surface clusters of sequence variation and potential differences in phosphorylation.

    Directory of Open Access Journals (Sweden)

    Dinesh C Soares

    2009-07-01

    Full Text Available Despite sharing 92% sequence identity, paralogous human translation elongation factor 1 alpha-1 (eEF1A1 and elongation factor 1 alpha-2 (eEF1A2 have different but overlapping functional profiles. This may reflect the differential requirements of the cell-types in which they are expressed and is consistent with complex roles for these proteins that extend beyond delivery of tRNA to the ribosome.To investigate the structural basis of these functional differences, we created and validated comparative three-dimensional (3-D models of eEF1A1 and eEF1A2 on the basis of the crystal structure of homologous eEF1A from yeast. The spatial location of amino acid residues that vary between the two proteins was thereby pinpointed, and their surface electrostatic and lipophilic properties were compared. None of the variations amongst buried amino acid residues are judged likely to have a major structural effect on the protein fold, or to affect domain-domain interactions. Nearly all the variant surface-exposed amino acid residues lie on one face of the protein, in two proximal but distinct sub-clusters. The result of previously performed mutagenesis in yeast may be interpreted as confirming the importance of one of these clusters in actin-bundling and filament disorganization. Interestingly, some variant residues lie in close proximity to, and in a few cases show differences in interactions with, residues previously inferred to be directly involved in binding GTP/GDP, eEF1Balpha and aminoacyl-tRNA. Additional sequence-based predictions, in conjunction with the 3-D models, reveal likely differences in phosphorylation sites that could reconcile some of the functional differences between the two proteins.The revelation and putative functional assignment of two distinct sub-clusters on the surface of the protein models should enable rational site-directed mutagenesis, including homologous reverse-substitution experiments, to map surface binding patches onto these

  1. piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART for genetic screens in mice.

    Directory of Open Access Journals (Sweden)

    Sean F Landrette

    Full Text Available Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.

  2. piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART) for genetic screens in mice.

    Science.gov (United States)

    Landrette, Sean F; Cornett, Jonathan C; Ni, Thomas K; Bosenberg, Marcus W; Xu, Tian

    2011-01-01

    Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.

  3. Use of a simian virus 40-based shuttle vector to analyze enhanced mutagenesis in mitomycin C-treated monkey cells

    International Nuclear Information System (INIS)

    Roilides, E.; Munson, P.J.; Levine, A.S.; Dixon, K.

    1988-01-01

    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

  4. Induced mutagenesis in dam- mutants of Escherichia coli: A role for 6-methyladenine residues in mutation avoidance

    International Nuclear Information System (INIS)

    Glickmann, B.; Elsen, P. van den; Radmann, M.

    1978-01-01

    E. coli strains carrying the dam-3 and dam-4 mutations resulting in reduced levels of 6-methyladenine in the DNA have been found to be more sensitive to base analogue mutagenesis than dam + strains. Mutagenesis by EMS was also found to be enhanced in dam - strains. Dam - mutants, however, were not found to be hypermutable by UV light. It is concluded that the dam - strains are deficient in the correct repair of mispairing lesions. The data are consistent with the hypothesis that 6-methyladenine residues in the DNA are involved in strand discrimination during mismatch correction. (orig.) [de

  5. A Mutagenesis Assay for Reporter Gene Screening Using Partially Degenerate Oligonucleotides of the Tandems NNT and NNC

    Directory of Open Access Journals (Sweden)

    Huifen Xu

    2015-01-01

    Full Text Available Not all proteins are tolerable to mutations. Whether a specific protein can be a mutable target is of importance in the biotechnology and pharmaceutical industry. This study reported a novel mutagenesis assay using tandem NNT and NNC oligonucleotides to test the mutability of a candidate gene. These two tandem oligonucleotides avoid the risk of forming nonsense mutations and render flexibility of truncating or expanding the insertion size. As a reporter gene, ZeoR (zeocin resistance gene was confirmed to have a high tolerance for mutagenesis by this new assay.

  6. Improvement of Alcaligenes faecalis nitrilase by gene site saturation mutagenesis and its application in stereospecific biosynthesis of (R)-(-)-mandelic acid.

    Science.gov (United States)

    Liu, Zhi-Qiang; Zhang, Xin-Hong; Xue, Ya-Ping; Xu, Ming; Zheng, Yu-Guo

    2014-05-21

    Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(-)-mandelic acid after 7.5 h of conversion reached 693 mM with an enantiomeric excess of 99%, and the space-time productivity of Mut3 was 21.50-fold higher than that of wild-type nitrilase. The Km, Vmax, and k(cat) of wild-type and Mut3 for mandelonitrile were 20.64 mM, 33.74 μmol mg(-1) min(-1), 24.45 s(-1), and 9.24 mM, 47.68 μmol mg(-1) min(-1), and 34.55 s(-1), respectively. A homology modeling and molecular docking study showed that the diameter of the catalytic tunnel of Mut3 became longer and that the tunnel volume was smaller. These structural changes are proposed to improve the hydrolytic activity and pH stability of Mut3. Mut3 has the potential for industrial applications in the upscale production of (R)-(-)-mandelic acid.

  7. Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System

    Directory of Open Access Journals (Sweden)

    Andrew R. Bassett

    2013-07-01

    Full Text Available Here, we present a simple and highly efficient method for generating and detecting mutations of any gene in Drosophila melanogaster through the use of the CRISPR/Cas9 system (clustered regularly interspaced palindromic repeats/CRISPR-associated. We show that injection of RNA into the Drosophila embryo can induce highly efficient mutagenesis of desired target genes in up to 88% of injected flies. These mutations can be transmitted through the germline to make stable lines. Our system provides at least a 10-fold improvement in efficiency over previously published reports, enabling wider application of this technique. We also describe a simple and highly sensitive method of detecting mutations in the target gene by high-resolution melt analysis and discuss how the new technology enables the study of gene function.

  8. Enu mutagenesis identifies a novel platelet phenotype in a loss-of-function Jak2 allele.

    Directory of Open Access Journals (Sweden)

    Nicole M Anderson

    Full Text Available Utilizing ENU mutagenesis, we identified a mutant mouse with elevated platelets. Genetic mapping localized the mutation to an interval on chromosome 19 that encodes the Jak2 tyrosine kinase. We identified a A3056T mutation resulting in a premature stop codon within exon 19 of Jak2 (Jak2(K915X, resulting in a protein truncation and functionally inactive enzyme. This novel platelet phenotype was also observed in mice bearing a hemizygous targeted disruption of the Jak2 locus (Jak2(+/-. Timed pregnancy experiments revealed that Jak2(K915X/K915X and Jak2(-/- displayed embryonic lethality; however, Jak2(K915X/K915X embryos were viable an additional two days compared to Jak2(-/- embryos. Our data suggest that perturbing JAK2 activation may have unexpected consequences in elevation of platelet number and correspondingly, important implications for treatment of hematological disorders with constitutive Jak2 activity.

  9. Random Transposon Mutagenesis for Cell-Envelope Resistant to Phage Infection.

    Science.gov (United States)

    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.

  10. Mutagenesis for ACMV resistance in a Ghanian cassava cultivar 'Bosom nsia'

    International Nuclear Information System (INIS)

    Ahiabu, R.K.; Lokko, Y.; Danso, K.; Klu, G.Y.P.

    1997-01-01

    Breeding for resistance to the African Cassava Mosaic Virus (ACMV) disease in the Ghanian cassava cultivar 'Bosom nsia' has been on-going for the past four years at the Biotechnology and Nuclear Agricultural Research Institute. Protocols for in vitro culture from shoot meristems and acclimation of plantlets were established. Radiosensitivity tests on the regenerated plantlets indicated LD 50 of 40 Gy, and doses of 25, 30 and 35 Gy were suitable for mutagenesis. These doses were applied to in vivo and in vitro grown plants, and selection was carried out in three propagations. Four variants, selected under field conditions with high viral incidence, were analyzed for virus particles with three virus indexing techniques. Polymerase chain reaction (PCR) product analysis of DNA extracts from Nicotiana benthamiana test plants, inoculated with sap from the leaves of variants, confirmed the presence of virus particles in all variants. Inoculation and ELISA tests suggested ACMV tolerance in selected variants. (author). 24 refs, 3 figs, 1 tab

  11. Mutagenesis Strategies for the Enhancement of Glucose Oxidase Production from Corn Steep Liquor

    International Nuclear Information System (INIS)

    Khalaf, M.A.

    2007-01-01

    During screening of the twenty eight Aspergillus and Penicillium species for GOD production, only A. niger (84) was observed to release high extra (3.15 U ml -1 ) and intracellular (9.30 U mg -l ) GOD activity. Conidia of A. niger S4 were subjected to mutagenesis with UV radiation, nitrous acid, and sodium azide, as a single or combined treatments, and GOD activity was detected with the diffusion plate method. Out of 27 over producing mutants tested in shaken flasks, UVNA54 mutant strain showed the highest level of GOD activity (171 %, higher than the wild type). Using CSL at concentration 25 ml rl as the sol nutrient source, the enzyme activity was increased to 5.16 U ml -1 and 22.40 U mg -1 for extra and intracellular, respectively. Viable cell numbers of Pseudomonas and Salmonella spp. decreased as the concentration of the produced enzyme increased from 1 to 3 U ml -1

  12. Artificial mutagenesis as an aid in overcoming genetic vulnerability of crop plants.

    Science.gov (United States)

    Konzak, C F; Nilan, R A; Kleinhofs, A

    Artificially induced genetic variation is being used effectively to supplement or complement sources of natural origin for practical plant breeding. Thus, creating genetic variation uill become increasingly important as crop genetic resources become more difficult to obtain via plant exploration. The aritificial induction of useful genetic variation offers important elements that can be used for overcoming genetic vulnerability: (1) new, previously unknown alleles can be induced in crop plant species to broaden the base of variation; (2) useful genetic variation can be induced in modern cultivars helping to shorten breeding time or to extend production "life"; (3) characteristics of existing genetic resource stocks can be improved to make them more useful in breeding; and (4) recombination in crosses may be enhanced. The performance of induced mutant crop cultivars and the successful uses of induced genetic variation in cross breeding indicate that artificial mutagenesis will play an increasingly greater role in plant breeding.

  13. Insertional mutagenesis reveals genes involved in Bacillus cereus ATCC 14579 growth at low temperature.

    Science.gov (United States)

    Broussolle, Véronique; Pandiani, Franck; Haddad, Nabila; Michaud, Caroline; Carlin, Frédéric; Nguyen-the, Christophe; Brillard, Julien

    2010-05-01

    Transposon mutagenesis of Bacillus cereus ATCC 14579 yielded cold-sensitive mutants. Mutants of genes encoding enzymes of the central metabolism were affected by cold, but also by other stresses, such as pH or salt, whereas a mutant with transposon insertion in the promoter region of BC0259 gene, encoding a putative DEAD-box RNA helicase displaying homology with Escherichia coli CsdA and Bacillus subtilis CshA RNA helicases, was only cold-sensitive. Expression of the BC0259 gene at 10 degrees C is reduced in the mutant. Analysis of the 5' untranslated region revealed the transcriptional start and putative cold shock-responsive elements. The role of this RNA helicase in the cold-adaptive response of B. cereus is discussed.

  14. Inducible pathway is required for mutagenesis in Salmonella typhimurium LT2

    International Nuclear Information System (INIS)

    Orrego, C.; Eisenstadt, E.

    1987-01-01

    UV mutability of Salmonella typhimurium LT2 was eliminated in the presence of a multicopy plasmid carrying the Escherichia coli lexA + gene. This result suggests that inducible, SOS-like functions are required for UV mutagenesis in S. typhimurium. S. typhimurium strains carrying either point or deletion mutations in topA had previously been shown to lose their mutability by UV or methyl methanesulfonate. Mitomycin C induction of the Phi(mucB'-lacZ') fusion (a DNA damage-inducible locus carried on plasmid pSE205) in S. typhimurium topA was normal, suggesting that RecA is activated in topA mutants. These observations lead the authors deduce that S. typhimurium has at least one DNA damage-inducible locus in addition to recA that is required for UV mutability

  15. Use of the radio mutagenesis gamma to diminish the germinative periods of the seeds

    International Nuclear Information System (INIS)

    Ramos Espinosa, K.A.; Damera Martinez, A.; Benito Zamora, E.; Vazquez Hernandez, L.

    2001-01-01

    Among the nuclear techniques that have been used in Cuba in to feasible way for their it lives it uses in the agriculture it dog think about that the radio mutagenesis is that of advance it lives. In this address the fundamental problem consists in that to the being to casuistically method forces to uses to great initial population. The Fruit Bomb Net Maradol was studied and it was obtained to diminish its germination period in 12 days without affecting the quality of the cultivation. The seeds were irradiated with an irradiator with source of Co-60, originator of radiations a (gamma) with to power of dose of 2,76 kGy/h and to half energy of 1,25 MeV. The dose range applied to the seeds was among 50 - 250 Gy

  16. Highly Efficient Site-Specific Mutagenesis in Malaria Mosquitoes Using CRISPR

    Directory of Open Access Journals (Sweden)

    Ming Li

    2018-02-01

    Full Text Available Anopheles mosquitoes transmit at least 200 million annual malaria infections worldwide. Despite considerable genomic resources, mechanistic understanding of biological processes in Anopheles has been hampered by a lack of tools for reverse genetics. Here, we report successful application of the CRISPR/Cas9 system for highly efficient, site-specific mutagenesis in the diverse malaria vectors Anopheles albimanus, A. coluzzii, and A. funestus. When guide RNAs (gRNAs and Cas9 protein are injected at high concentration, germline mutations are common and usually biallelic, allowing for the rapid creation of stable mutant lines for reverse genetic analysis. Our protocol should enable researchers to dissect the molecular and cellular basis of anopheline traits critical to successful disease transmission, potentially exposing new targets for malaria control.

  17. Agrobacterium tumefaciens-mediated transformation: An efficient tool for insertional mutagenesis and targeted gene disruption in Harpophora oryzae.

    Science.gov (United States)

    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. Copyright © 2015 Elsevier GmbH. All rights reserved.

  18. Codon cassette mutagenesis: a general method to insert or replace individual codons by using universal mutagenic cassettes.

    Science.gov (United States)

    Kegler-Ebo, D M; Docktor, C M; DiMaio, D

    1994-05-11

    We describe codon cassette mutagenesis, a simple method of mutagenesis that uses universal mutagenic cassettes to deposit single codons at specific sites in double-stranded DNA. A target molecule is first constructed that contains a blunt, double-strand break at the site targeted for mutagenesis. A double-stranded mutagenic codon cassette is then inserted at the target site. Each mutagenic codon cassette contains a three base pair direct terminal repeat and two head-to-head recognition sequences for the restriction endonuclease Sapl, an enzyme that cleaves outside of its recognition sequence. The intermediate molecule containing the mutagenic cassette is then digested with Sapl, thereby removing most of the mutagenic cassette, leaving only a three base cohesive overhang that is ligated to generate the final insertion or substitution mutation. A general method for constructing blunt-end target molecules suitable for this approach is also described. Because the mutagenic cassette is excised during this procedure and alters the target only by introducing the desired mutation, the same cassette can be used to introduce a particular codon at all target sites. Each cassette can deposit two different codons, depending on the orientation in which it is inserted into the target molecule. Therefore, a series of eleven cassettes is sufficient to insert all possible amino acids at any constructed target site. Thus codon cassettes are 'off-the-shelf' reagents, and this methodology should be a particularly useful and inexpensive approach for subjecting multiple different positions in a protein sequence to saturation mutagenesis.

  19. RecA-mediated cleavage activates UmuD for mutagenesis: Mechanistic relationship between transcriptional derepression and posttranslational activation

    International Nuclear Information System (INIS)

    Nohmi, Takehiko; Battista, J.R.; Dodson, L.A.; Walker, G.C.

    1988-01-01

    The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. It has been shown that the UmuD protein shares homology with LexA, the repressor of the SOS genes. In this paper the authors describe a series of genetic experiments that indicate that the purpose of RecA-mediated cleavage of UmuD at its bond between Cys-24 and Gly-25 is to activate UmuD for its role in mutagenesis and that the COOH-terminal fragment of UmuD is necessary and sufficient for the role of UmuD in UV mutagenesis. Other genetic experiments are presented that (i) support the hypothesis that the primary role of Ser-60 in UmuD function is to act as a nucleophile in the RecA-mediated cleavage reaction and (ii) raise the possibility that RecA has a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD

  20. [Comparative mutagenesis of human cells in vivo and in vitro]. Progress report, January 1-December 30, 1985

    International Nuclear Information System (INIS)

    1985-01-01

    Annual progress report is made on project focusing on the comparative mutagenesis of human cells in vivo and in vitro. The study employs the HGPRT gene to explore the changes in nucleotide sequence which has occurred in spontaneous mutations or mutations induced by MNNG or ICR191. Reports on the individual projects have been abstracted and indexed for the Energy Data Base. (DT)

  1. Effect of radiation-sensitive mutations and mutagens/carcinogens on bacterial recombination and mutagenesis. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Matney, T.S.

    1978-01-01

    Progress is reported on effects of temperature sensitive DNA-initiation mutation in E. coli K-12 mutants; the use of Bacillus subtilis transforming system as an in vitro mutagenesis system; characteristics of the E. coli lysogen used to test the permeability to polycyclic aromatic hydrocarbons; and the genetic toxicology of gentian violet. (PCS)

  2. Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation

    DEFF Research Database (Denmark)

    Vernet, Erik; Sauer, Jørgen; Andersen, Peter Andreas

    2011-01-01

    Ligation-independent cloning (LIC) allows for cloning of DNA constructs independent of insert restriction sites and ligases. However, any required mutations are typically introduced by additional, time-consuming steps. We present a rapid, inexpensive method for mutagenesis in the 5' LIC site of e...

  3. Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus.

    Directory of Open Access Journals (Sweden)

    Gabriel Rinaldi

    Full Text Available Functional studies will facilitate characterization of role and essentiality of newly available genome sequences of the human schistosomes, Schistosoma mansoni, S. japonicum and S. haematobium. To develop transgenesis as a functional approach for these pathogens, we previously demonstrated that pseudotyped murine leukemia virus (MLV can transduce schistosomes leading to chromosomal integration of reporter transgenes and short hairpin RNA cassettes. Here we investigated vertical transmission of transgenes through the developmental cycle of S. mansoni after introducing transgenes into eggs. Although MLV infection of schistosome eggs from mouse livers was efficient in terms of snail infectivity, >10-fold higher transgene copy numbers were detected in cercariae derived from in vitro laid eggs (IVLE. After infecting snails with miracidia from eggs transduced by MLV, sequencing of genomic DNA from cercariae released from the snails also revealed the presence of transgenes, demonstrating that transgenes had been transmitted through the asexual developmental cycle, and thereby confirming germline transgenesis. High-throughput sequencing of genomic DNA from schistosome populations exposed to MLV mapped widespread and random insertion of transgenes throughout the genome, along each of the autosomes and sex chromosomes, validating the utility of this approach for insertional mutagenesis. In addition, the germline-transmitted transgene encoding neomycin phosphotransferase rescued cultured schistosomules from toxicity of the antibiotic G418, and PCR analysis of eggs resulting from sexual reproduction of the transgenic worms in mice confirmed that retroviral transgenes were transmitted to the next (F1 generation. These findings provide the first description of wide-scale, random insertional mutagenesis of chromosomes and of germline transmission of a transgene in schistosomes. Transgenic lines of schistosomes expressing antibiotic resistance could advance

  4. Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis.

    Science.gov (United States)

    Castro-Cerritos, Karla Viridiana; Yasbin, Ronald E; Robleto, Eduardo A; Pedraza-Reyes, Mario

    2017-02-15

    The Gram-positive microorganism Bacillus subtilis relies on a single class Ib ribonucleotide reductase (RNR) to generate 2'-deoxyribonucleotides (dNDPs) for DNA replication and repair. In this work, we investigated the influence of RNR levels on B. subtilis stationary-phase-associated mutagenesis (SPM). Since RNR is essential in this bacterium, we engineered a conditional mutant of strain B. subtilis YB955 (hisC952 metB5 leu427) in which expression of the nrdEF operon was modulated by isopropyl-β-d-thiogalactopyranoside (IPTG). Moreover, genetic inactivation of ytcG, predicted to encode a repressor (NrdR) of nrdEF in this strain, dramatically increased the expression levels of a transcriptional nrdE-lacZ fusion. The frequencies of mutations conferring amino acid prototrophy in three genes were measured in cultures under conditions that repressed or induced RNR-encoding genes. The results revealed that RNR was necessary for SPM and overexpression of nrdEF promoted growth-dependent mutagenesis and SPM. We also found that nrdEF expression was induced by H 2 O 2 and such induction was dependent on the master regulator PerR. These observations strongly suggest that the metabolic conditions operating in starved B. subtilis cells increase the levels of RNR, which have a direct impact on SPM. Results presented in this study support the concept that the adverse metabolic conditions prevailing in nutritionally stressed bacteria activate an oxidative stress response that disturbs ribonucleotide reductase (RNR) levels. Such an alteration of RNR levels promotes mutagenic events that allow Bacillus subtilis to escape from growth-limited conditions. Copyright © 2017 American Society for Microbiology.

  5. Mutagenesis in sequence encoding of human factor VII for gene therapy of hemophilia

    Directory of Open Access Journals (Sweden)

    B Kazemi

    2009-12-01

    Full Text Available "nBackground: Current treatment of hemophilia which is one of the most common bleeding disorders, involves replacement therapy using concentrates of FVIII and FIX .However, these concentrates have been associated with viral infections and thromboembolic complications and development of antibodies. "nThe use of recombinant human factor VII (rhFVII is effective  for the treatment of patients with  hemophilia A or B, who develop antibodies ( referred as inhibitors against  replacement therapy , because it induces coagulation independent of FVIII and FIX. However, its short half-life and high cost have limited its use. One potential solution to this problem may be the use of FVIIa gene transfer, which would attain continuing therapeutic levels of expression from a single injection. The aim of this study was to engineer a novel hFVII (human FVII gene containing a cleavage site for the intracellular protease and furin, by PCR mutagenesis "nMethods: The sequence encoding light and heavy chains of hFVII, were amplified by using hFVII/pTZ57R and specific primers, separately. The PCR products were cloned in pTZ57R vector. "nResults and discussion: Cloning was confirmed by restriction analysis or PCR amplification using specific primers and plasmid universal primers. Mutagenesis of sequence encoding light and heavy chain was confirmed by restriction enzyme. "nConclusion: In the present study, it was provided recombinant plasmids based on mutant form of DNA encoding light and heavy chains.  Joining mutant form of DNA encoding light chain with mutant heavy chain led to a new variant of hFVII. This variant can be activated by furin and an increase in the proportion of activated form of FVII. This mutant form of hFVII may be used for gene therapy of hemophilia.

  6. Rolling Circle Mutagenesis of GST-mCherry to Understand Mutation, Gene Expression, and Regulation

    Directory of Open Access Journals (Sweden)

    Jessica Cole

    2017-05-01

    Full Text Available Undergraduates are often familiar with textbook examples of human mutations that affect coding regions and the subsequent disorders, but they may struggle with understanding the implications of mutations in the regulatory regions of genes. We have designed a laboratory sequence that will allow students to explore the effect random mutagenesis can have on protein function, expression, and ultimately phenotype. Students design and perform a safe and time-efficient random mutagenesis experiment using error-prone rolling circular amplification of a plasmid expressing the inducible fusion protein glutathione S-transferase (GST-mCherry. Mutagenized and wild-type control plasmid DNA, respectively, are then purified and transformed into bacteria to assess phenotypic changes. While bacteria transformed with the wild type control should be pink, some bacterial colonies transformed with mutagenized plasmids will exhibit a different color. Students attempt to identify their mutations by isolating plasmid from these mutant colonies, sequencing, and comparing their mutant sequence to the wild-type sequence. Additionally, students evaluate the potential effects of mutations on protein production by inducing GST-mCherry expression in cultures, generating cell lysates, and analyzing them using SDS-PAGE. Students who have a phenotypic difference but do not obtain a coding region mutation will be able to think critically about plasmid structure and regulation outside of the gene sequence. Students who do not obtain bacterial transformants have the chance to contemplate how mutation of antibiotic resistance genes or replication origins may have contributed to their results. Overall, this series of laboratories exposes students to basic genetic techniques and helps them conceptualize mutation beyond coding regions.

  7. Improving the chitinolytic activity of Bacillus pumilus SG2 by random mutagenesis.

    Science.gov (United States)

    Vahed, Majid; Motalebi, Ebrahim; Rigi, Garshasb; Akbari Noghabi, Kambiz; Soudi, Mohammad Reza; Sadeghi, Mehdi; Ahmadian, Gholamreza

    2013-11-28

    Bacillus pumilus SG2, a halotolerant strain, expresses two major chitinases designated ChiS and ChiL that were induced by chitin and secreted into the supernatant. The present work aimed to obtain a mutant with higher chitinolytic activity through mutagenesis of Bacillus pumilus SG2 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on chitin agar and subsequent formation of halos, the mutated strains were examined for degradation of chitin under different conditions. A mutant designated AV2-9 was selected owing to its higher chitinase activity. To search for possible mutations in the whole operon including ChiS and ChiL, the entire chitinase operon, including the intergenic region, promoter, and two areas corresponding to the ChiS and ChiL ORF, was suquenced. Nucleotide sequence analysis of the complete chitinase operon from the SG2 and AV2-9 strains showed the presence of a mutation in the catalytic domain (GH18) of chitinase (ChiL). The results demonstrated that a single base change had occurred in the ChiL sequence in AV2- 9. The wild-type chitinase, ChiL, and the mutant (designated ChiLm) were cloned, expressed, and purified in E. coli. Both enzymes showed similar profiles of activity at different ranges of pH, NaCl concentration, and temperature, but the mutant enzyme showed approximately 30% higher catalytic activity under all the conditions tested. The results obtained in this study showed that the thermal stability of chitinase increased in the mutant strain. Bioinformatics analysis was performed to predict changes in the stability of proteins caused by mutation.

  8. Predicting resistance by mutagenesis: lessons from 45 years of MBC resistance

    Directory of Open Access Journals (Sweden)

    Nichola J. Hawkins

    2016-11-01

    Full Text Available When a new fungicide class is introduced, it is useful to anticipate the resistance risk in advance, attempting to predict both risk level and potential mechanisms. One tool for the prediction of resistance risk is laboratory selection for resistance, with the mutational supply increased through UV or chemical mutagenesis. This enables resistance to emerge more rapidly than in the field, but may produce mutations that would not emerge under field conditions.The methyl-benzimidazole carbamates (MBCs were the first systemic single-site agricultural fungicides, and the first fungicides affected by rapid evolution of target-site resistance. MBC resistance has now been reported in over 90 plant pathogens in the field, and laboratory mutants have been studied in nearly 30 species.The most common field mutations, including β-tubulin E198A/K/G, F200Y and L240F, have all been identified in laboratory mutants. However, of 28 mutations identified in laboratory mutants, only nine have been reported in the field. Therefore, the predictive value of mutagenesis studies would be increased by understanding which mutations are likely to emerge in the field.Our review of the literature indicates that mutations with high resistance factors, and those found in multiple species, are more likely to be reported in the field. However, there are many exceptions, possibly due to fitness penalties. Whether a mutation occurred in the same species appears less relevant, perhaps because β-tubulin is highly conserved so functional constraints are similar across all species. Predictability of mutations in other target sites will depend on the level and conservation of constraints.

  9. DNA-reactive protein monoepoxides induce cell death and mutagenesis in mammalian cells.

    Science.gov (United States)

    Tretyakova, Natalia Y; Michaelson-Richie, Erin D; Gherezghiher, Teshome B; Kurtz, Jamie; Ming, Xun; Wickramaratne, Susith; Campion, Melissa; Kanugula, Sreenivas; Pegg, Anthony E; Campbell, Colin

    2013-05-07

    Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to act by cross-linking cellular biomolecules, their exact mechanisms of action have not been established. In cells, these compounds form a mixture of DNA lesions, including nucleobase monoadducts, interstrand and intrastrand cross-links, and DNA-protein cross-links (DPCs). Interstrand DNA-DNA cross-links block replication and transcription by preventing DNA strand separation, contributing to toxicity and mutagenesis. In contrast, potential contributions of drug-induced DPCs are poorly understood. To gain insight into the biological consequences of DPC formation, we generated DNA-reactive protein reagents and examined their toxicity and mutagenesis in mammalian cells. Recombinant human O(6)-alkylguanine DNA alkyltransferase (AGT) protein or its variants (C145A and K125L) were treated with 1,2,3,4-diepoxybutane to yield proteins containing 2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and mass spectrometry experiments confirmed that epoxide-functionalized AGT proteins formed covalent DPC but no other types of nucleobase damage when incubated with duplex DNA. Introduction of purified AGT monoepoxides into mammalian cells via electroporation generated AGT-DNA cross-links and induced cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene. Smaller numbers of DPC lesions and reduced levels of cell death were observed when using protein monoepoxides generated from an AGT variant that fails to accumulate in the cell nucleus (K125L), suggesting that nuclear DNA damage is required for toxicity. Taken together, these results indicate that AGT protein monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal DNA. More generally, these data suggest that covalent DPC lesions contribute to the cytotoxic and mutagenic effects of bis-electrophiles.

  10. Nutritional value of quinua (chenopodium quinoa) seeds improved by radioinduced mutagenesis

    International Nuclear Information System (INIS)

    De La Cruz, T. E.; Avila R, S.; Garcia R, A.

    2006-01-01

    Quinua (Chenopodium quinoa), a pseudo cereal considered as an alternative crop for areas with marginal agricultural conditions has been subjected since 1990 to a radioinduced mutagenesis programme aiming to obtain lines with low saponin content, good yields and high nutritional value. Seeds obtained from lines grown in M7 generation which exhibited yields averaging 1.5 ton/ha, were analyzed regarding grain quality and nutritive value. Evaluated parameters were diameter and thickness of the seed, weight of 100 seeds and density. Regarding to bromatological analysis, determinations were made of moisture, ash, raw fiber, proteins, oil content and carbohydrates, following the procedures indicated in Official Mexican Norms (NOM). Evaluated genotypes were the varieties Sajama, Barandales and Amarilla de Marangani and the mutant lines 20R110, 94, 20R333, 20R227, 20R342, 20R37 and the advanced line obtained by selection 640304. Mutant genotypes 20R333 and 20R342 exhibited outstanding characteristics regarding to grain quality (diameter 2.0 mm, thickness 1.2mm, weight of 100 seeds 0.42 and 0.22 g respectively and density 710 and 686 grams per liter). In the bromatological analysis the protein content ranged from 11.82 % (genotype 20 R227) to 16.8% in mutant 20R333 while mutant 20R342 exhibited 15.6%. The lipid content was minimum on Barandales and 20R333 both with 3.8%, having the genotype 20R110 the highest value among evaluated genotypes with 4.35%, line 20R342 exhibited 4.2%.. The high percentages of proteins and lipids, found among some analyzed mutants exhibit the feasibility to obtain, through radioinduced mutagenesis, lines with low saponins and high nutritive value

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

    Science.gov (United States)

    Maisnier-Patin, Sophie; Roth, John R.

    2015-01-01

    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. PMID:26134316

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

    Science.gov (United States)

    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. Copyright © 2015 Cold

  13. NHEJ enzymes LigD and Ku participate in stationary-phase mutagenesis in Pseudomonas putida.

    Science.gov (United States)

    Paris, Ülvi; Mikkel, Katren; Tavita, Kairi; Saumaa, Signe; Teras, Riho; Kivisaar, Maia

    2015-07-01

    Under growth-restricting conditions bacterial populations can rapidly evolve by a process known as stationary-phase mutagenesis. Bacterial nonhomologous end-joining (NHEJ) system which consists of the DNA-end-binding enzyme Ku and the multifunctional DNA ligase LigD has been shown to be important for survival of bacteria especially during quiescent states, such as late stationary-phase populations or sporulation. In this study we provide genetic evidence that NHEJ enzymes participate in stationary-phase mutagenesis in a population of carbon-starved Pseudomonas putida. Both the absence of LigD or Ku resulted in characteristic spectra of stationary-phase mutations that differed from each other and also from the wild-type spectrum. This indicates that LigD and Ku may participate also in mutagenic pathways that are independent from each other. Our results also imply that both phosphoesterase (PE) and polymerase (POL) domains of the LigD protein are involved in the occurrence of mutations in starving P. putida. The participation of both Ku and LigD in the occurrence of stationary-phase mutations was further supported by the results of the analysis of mutation spectra in stationary-phase sigma factor RpoS-minus background. The spectra of mutations identified in the RpoS-minus background were also distinct if LigD or Ku was absent. Interestingly, the effects of the presence of these enzymes on the frequency of occurrence of certain types of mutations were different or even opposite in the RpoS-proficient and deficient backgrounds. These results imply that RpoS affects performance of mutagenic pathways in starving P. putida that utilize LigD and/or Ku. Copyright © 2015. Published by Elsevier B.V.

  14. In vivo expression technology and signature-tagged mutagenesis screens for identifying mechanisms of survival of zoonotic foodborne pathogens.

    Science.gov (United States)

    Dudley, Edward G

    2008-08-01

    High-throughput genetic screens provide great insights into the biochemistry and molecular biology of how bacteria sense, respond to, and propagate within their environments. Genomics era techniques such as microarrays and proteomics have great potential to increase our understanding of how foodborne pathogens grow and survive within animal and human hosts, in the environment and foods, and during thermal and nonthermal inactivation protocols. While these techniques are incredibly useful for studying gene expression in simplified in vitro conditions, it is much more challenging to pursue similar studies within more complex experimental models such as in vivo, within the food matrix, or within heterogeneous microbial populations. Techniques such as in vivo expression technology (IVET) and signature-tagged mutagenesis (STM) provide alternatives for studying bacterial gene expression and growth requirements within these settings. These techniques are used extensively by the medical, veterinary, and plant research communities for identifying genes promoting the colonization and disease process, factors mediating commensalism between bacteria and their host, and genes that promote survival of environmental bacteria within natural settings. Research into the transmission and survival of foodborne pathogens from farm-to-fork would likely benefit from these techniques, however there are few reports describing their use for such purposes. This review will briefly cover the methods of IVET and STM, discuss how these techniques improved our understanding of the interactions between zoonotic foodborne pathogens and their animal hosts, and ask whether these techniques could be further exploited to better understand the survival of foodborne pathogens within the environment, within food matrices, and during inactivation protocols.

  15. Screening a random mutagenesis library of a fungal β-fructofuranosidase using FT-MIR ATR spectroscopy and multivariate analysis.

    Science.gov (United States)

    Trollope, K M; Nieuwoudt, H H; Görgens, J F; Volschenk, H

    2014-05-01

    Short-chain fructooligosaccharides (scFOS) are valuable health-promoting food additives. During the batch production of scFOS from sucrose the β-fructofuranosidase catalyst is subject to product inhibition by glucose. Engineering the enzyme for reduced sensitivity to glucose could improve product yields or process productivity while preserving the simple industrial batch design. Random mutagenesis is a useful technique for engineering proteins but should be coupled to a relevant high-throughput screen. Such a screen for sucrose and scFOS quantification remains elusive. This work presents the development of a screening method displaying potential high-throughput capacity for the evaluation of β-fructofuranosidase libraries using Fourier transform mid-infrared attenuated total reflectance (FT-MIR ATR) spectroscopy and multivariate analysis. A calibration model for the quantification of sucrose in enzyme assay samples ranged from 5 to 200 g/l and the standard error of prediction was below 13 g/l. A library of the Aspergillus japonicus fopA gene was generated by error prone PCR and screened in Saccharomyces cerevisiae. Using FT-MIR ATR spectroscopy, potential hits were identified as those variants that converted more sucrose in the presence of the glucose inhibitor than the parent. Subsequent analysis of reaction products generated by top performers using high-performance liquid chromatography identified a variant producing higher scFOS levels than the parent. At the peak difference in performance the variant produced 28 % more scFOS from the same amount of sucrose. This study highlights the application of FT-MIR ATR spectroscopy to a variant discovery pipeline in the directed evolution of a β-fructofuranosidase for enhanced scFOS production.

  16. Protein engineering by random mutagenesis and structure-guided consensus of Geobacillus stearothermophilus Lipase T6 for enhanced stability in methanol.

    Science.gov (United States)

    Dror, Adi; Shemesh, Einav; Dayan, Natali; Fishman, Ayelet

    2014-02-01

    The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production.

  17. Efficient methods for targeted mutagenesis in zebrafish using zinc-finger nucleases: data from targeting of nine genes using CompoZr or CoDA ZFNs.

    Directory of Open Access Journals (Sweden)

    Raman Sood

    Full Text Available Recently, it has been shown that targeted mutagenesis using zinc-finger nucleases (ZFNs and transcription activator-like effector nucleases (TALENs can be used to generate knockout zebrafish lines for analysis of their function and/or developing disease models. A number of different methods have been developed for the design and assembly of gene-specific ZFNs and TALENs, making them easily available to most zebrafish researchers. Regardless of the choice of targeting nuclease, the process of generating mutant fish is similar. It is a time-consuming and multi-step process that can benefit significantly from development of efficient high throughput methods. In this study, we used ZFNs assembled through either the CompoZr (Sigma-Aldrich or the CoDA (context-dependent assembly platforms to generate mutant zebrafish for nine genes. We report our improved high throughput methods for 1 evaluation of ZFNs activity by somatic lesion analysis using colony PCR, eliminating the need for plasmid DNA extractions from a large number of clones, and 2 a sensitive founder screening strategy using fluorescent PCR with PIG-tailed primers that eliminates the stutter bands and accurately identifies even single nucleotide insertions and deletions. Using these protocols, we have generated multiple mutant alleles for seven genes, five of which were targeted with CompoZr ZFNs and two with CoDA ZFNs. Our data also revealed that at least five-fold higher mRNA dose was required to achieve mutagenesis with CoDA ZFNs than with CompoZr ZFNs, and their somatic lesion frequency was lower (<5% when compared to CopmoZr ZFNs (9-98%. This work provides high throughput protocols for efficient generation of zebrafish mutants using ZFNs and TALENs.

  18. Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

    Science.gov (United States)

    Dror, Adi; Shemesh, Einav; Dayan, Natali

    2014-01-01

    The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production. PMID:24362426

  19. Identification of a Proton-Chloride Antiporter (EriC) by Himar1 Transposon Mutagenesis in Lactobacillus reuteri and Its Role in Histamine Production

    Science.gov (United States)

    Hemarajata, P; Spinler, JK; Balderas, MA; Versalovic, J

    2014-01-01

    The gut microbiome may modulate intestinal immunity by luminal conversion of dietary amino acids to biologically active signals. The model probiotic organism Lactobacillus reuteri ATCC PTA 6475 is indigenous to the human microbiome, and converts the amino acid L-histidine to the biogenic amine, histamine. Histamine suppresses TNF production by human myeloid cells and is a product of L-histidine decarboxylation, which is a proton-facilitated reaction. A transposon mutagenesis strategy was developed based on a single-plasmid nisin-inducible Himar1 transposase/transposon delivery system for L. reuteri. A highly conserved proton-chloride antiporter gene (eriC), a gene widely present in the gut microbiome was discovered by Himar1 transposon (Tn)-mutagenesis presented in this study. Genetic inactivation of eriC by transposon insertion and genetic recombineering resulted in reduced ability of L. reuteri to inhibit TNF production by activated human myeloid cells, diminished histamine production by the bacteria and downregulated expression of histidine decarboxylase (hdc) cluster genes compared to those of WT 6475. EriC belongs to a large family of ion transporters that includes chloride channels and proton-chloride antiporters and may facilitate the availability of protons for the decarboxylation reaction, resulting in histamine production by L. reuteri. This report leverages the tools of bacterial genetics for probiotic gene discovery. The findings highlight the widely conserved nature of ion transporters in bacteria and how ion transporters are coupled with amino acid decarboxylation and contributed to microbiome-mediated immunomodulation. PMID:24488273

  20. Different efficiency of UmuDC and MucAB proteins in UV light induced mutagenesis in Escherichia coli

    International Nuclear Information System (INIS)

    Blanco, M.; Herrera, G.; Aleixandre, V.

    1986-01-01

    Two multicopy plasmids carrying either the umuDC or the mucAB operon were used to compare the efficiency of UmuDC and MucAB proteins in UV mutagenesis of Escherichia coli K12. It was found that in recA + uvr + bacteria, plasmid pIC80, mucAB + mediated UV mutagenesis more efficiently than did plasmid pSE 117, umuDC + . A similar result was obtained in lex A51(Def) cells, excluding the possibility that this was due to a differential regulation by LexA of the umuDC and mucAB operons. We conclude that some structural characteristic of the UmuDC and MucAB proteins determines their different efficiency in UV mutagenesis. This characteristic could be also responsible for the observation that in the recA430 mutant, pIC80 but no pSE117 can mediate UV mutagenesis. In the recAS142 mutant pIC80 also promoted UV mutagenesis more efficiently than pSE117. In this mutant, the recombination proficiency, the protease activity toward LexA and the mutation frequency were increased by the presence of adenine in the medium. In recA + uvrB5 bacteria, plasmid pSE117, umuDC caused both an increase in UV sensitivity as well as a reduction in the mutation frequency. These negative effects resulting from the overproduction of UmuDC proteins were higher in recA142 uvrB5 than in recA + uvrB5 cells. In contrast, overproduction of MucAB proteins in excision-deficient bacteria containing pIC80 led to a large increase in the mutation frequency. We suggest that the functional differences between UmuDC and MucAB proteins might be due to their different dependence on the direct role of RecA protease in UV mutagenesis. (orig.)

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

    Science.gov (United States)

    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. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. Mutagenesis of NosM Leader Peptide Reveals Important Elements in Nosiheptide Biosynthesis

    Science.gov (United States)

    Jin, Liang; Wu, Xuri; Xue, Yanjiu; Jin, Yue; Wang, Shuzhen

    2016-01-01

    ABSTRACT Nosiheptide, a typical member of the ribosomally synthesized and posttranslationally modified peptides (RiPPs), exhibits potent activity against multidrug-resistant Gram-positive bacterial pathogens. The precursor peptide of nosiheptide (NosM) is comprised of a leader peptide with 37 amino acids and a core peptide containing 13 amino acids. To pinpoint elements in the leader peptide that are essential for nosiheptide biosynthesis, a collection of mutants with unique sequence features, including N- and C-terminal motifs, peptide length, and specific sites in the leader peptide, was generated by mutagenesis in vivo. The effects of various mutants on nosiheptide biosynthesis were evaluated. In addition to the necessity of a conserved motif LEIS box, native length and the N-terminal 12 amino acid residues were indispensable, and single-site substitutions of these 12 amino acid residues resulted in changes ranging from a greater-than-5-fold decrease to a 2-fold increase of nosiheptide production, depending on the sites and substituted residues. Moreover, although the C-terminal motif is not conservative, significant effects of this portion on nosiheptide production were also evident. Taken together, the present results further highlight the importance of the leader peptide in nosiheptide biosynthesis, and provide new insights into the diversity and specificity of leader peptides in the biosynthesis of various RiPPs. IMPORTANCE As a representative thiopeptide, nosiheptide exhibits excellent antibacterial activity. Although the biosynthetic gene cluster and several modification steps have been revealed, the presence and roles of the leader peptide within the precursor peptide of the nosiheptide gene cluster remain elusive. Thus, identification of specific elements in the leader peptide can significantly facilitate the genetic manipulation of the gene cluster for increasing nosiheptide production or generating diverse analogues. Given the complexity of the

  3. Probing the interaction of brain fatty acid binding protein (B-FABP with model membranes.

    Directory of Open Access Journals (Sweden)

    Fábio Dyszy

    Full Text Available Brain fatty acid-binding protein (B-FABP interacts with biological membranes and delivers polyunsaturated fatty acids (FAs via a collisional mechanism. The binding of FAs in the protein and the interaction with membranes involve a motif called "portal region", formed by two small α-helices, A1 and A2, connected by a loop. We used a combination of site-directed mutagenesis and electron spin resonance to probe the changes in the protein and in the membrane model induced by their interaction. Spin labeled B-FABP mutants and lipidic spin probes incorporated into a membrane model confirmed that B-FABP interacts with micelles through the portal region and led to structural changes in the protein as well in the micelles. These changes were greater in the presence of LPG when compared to the LPC models. ESR spectra of B-FABP labeled mutants showed the presence of two groups of residues that responded to the presence of micelles in opposite ways. In the presence of lysophospholipids, group I of residues, whose side chains point outwards from the contact region between the helices, had their mobility decreased in an environment of lower polarity when compared to the same residues in solution. The second group, composed by residues with side chains situated at the interface between the α-helices, experienced an increase in mobility in the presence of the model membranes. These modifications in the ESR spectra of B-FABP mutants are compatible with a less ordered structure of the portal region inner residues (group II that is likely to facilitate the delivery of FAs to target membranes. On the other hand, residues in group I and micelle components have their mobilities decreased probably as a result of the formation of a collisional complex. Our results bring new insights for the understanding of the gating and delivery mechanisms of FABPs.

  4. Effects of harman and norharman on spontaneous and ultraviolet light-induced mutagenesis in cultured Chinese hamster cells

    International Nuclear Information System (INIS)

    Chang, C.C.; Castellazzi, M.; Glover, T.W.; Trosko, J.E.

    1978-01-01

    Nontoxic concentrations of harman and norharman were tested in cultured Chinese hamster cells for their effects on DNA repair and mutagenesis. The following effects of harman were observed: (a) the survival of ultraviolet light- or x-ray-damaged cells was reduced; (b) the ultraviolet light-induced unscheduled DNA synthesis was slightly inhibited; and (c) the frequency of spontaneous or ultraviolet light-induced ouabain-resistant (ouar) or 6-thioguanine-resistant (6-TGr) mutations was reduced. Furthermore, the effect of harman on survival and mutagenesis was greater than that of norharman and was detected primarily in treatments in which cells were exposed to harman immediately following ultraviolet light irradiation. Our data clearly indicate that harman decreases the capacity to repair DNA damage and fix mutations in Chinese hamster cells, possibly because of the intercalation properties of this compound

  5. Selection of Zygosaccharomyces rouxii strains resistant to cadmium with improved removal abilities through ultraviolet-diethyl sulfate cooperative mutagenesis.

    Science.gov (United States)

    Liu, Yu; Xu, Ying; Wang, Dongfeng; Jiang, Wei

    2017-08-01

    Cd 2+ resistance and bioaccumulation capacity were selected from parental Zygosaccharomyces rouxii (CRZ-0) while maintaining NaCl tolerance using protoplast mutagenesis technology. Ultraviolet-diethyl sulfate (UV-DES) cooperative mutagenesis, followed by preliminary screening and rescreening, was used to select the mutant strain CRZ-9. CRZ-9 grew better than CRZ-0 in YPD medium with 20 or 50 mg L -1 of Cd 2+ . Scanning electron microscopy observations and flow cytometry tests indicated that CRZ-9 was more effective at eliminating reactive oxygen species (ROS) generated by Cd 2+ , which led to less cellular structural damage and lower lethality. Furthermore, compared with CRZ-0, CRZ-9 exhibited increased potential for application with higher Cd 2+ removal ratio, wider working pH range, and lower biomass dosage in Cd 2+ bioaccumulation. The mutant strain CRZ-9 possessed improved Cd 2+ resistance and bioaccumulation capacity and therefore is a promising strain to remove Cd 2+ from wastewater.

  6. Enhanced vanillin production from recombinant E. coli using NTG mutagenesis and adsorbent resin.

    Science.gov (United States)

    Yoon, Sang-Hwal; Lee, Eun-Gyeong; Das, Amitabha; Lee, Sook-Hee; Li, Cui; Ryu, Hee-Kyoung; Choi, Myung-Suk; Seo, Weon-Taek; Kim, Seon-Won

    2007-01-01

    Vanillin production was tested with different concentrations of added ferulic acid in E. coli harboring plasmid pTAHEF containing fcs (feruloyl-CoA synthase) and ech (enoyl-CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104. The maximum production of vanillin from E. coli DH5alpha harboring pTAHEF was found to be 1.0 g/L at 2.0 g/L of ferulic acid for 48 h of culture. To improve the vanillin production by reducing its toxicity, two approaches were followed: (1) generation of vanillin-resistant mutant of NTG-VR1 through NTG mutagenesis and (2) removal of toxic vanillin from the medium by XAD-2 resin absorption. The vanillin production of NTG-VR1 increased to three times at 5 g/L of ferulic acid when compared with its wild-type strain. When 50% (w/v) of XAD-2 resin was employed in culture with 10 g/L of ferulic acid, the vanillin production of NTG-VR1 was 2.9 g/L, which was 2-fold higher than that obtained with no use of the resin.

  7. Mechanisms of mutagenesis in human cells exposed to 55 MeV protons

    Science.gov (United States)

    Gauny, S.; Wiese, C.; Kronenberg, A.

    2001-01-01

    Protons represent the major type of charged particle radiation in spaceflight environments. The purpose of this study was to assess mutations arising in human lymphoid cells exposed to protons. Mutations were quantitated at the thymidine kinase (TK1) locus in cell lines derived from the same donor: TK6 cells (wt TP53) and WTK1 cells (mutant TP53). WTK1 cells were much more susceptible to mutagenesis following proton exposure than TK6 cells. Intragenic deletions were observed among early-arising TK1 mutants in TK6 cells, but not in WTK1 cells where all of the mutants arose by LOH. Deletion was the predominant mode of LOH in TK6 cells, while allelic recombination was the major mode of LOH in WTK1 cells. Deletions were of variable lengths, from recombination often extended to the telomere. In summary, proton exposures elicited many types of mutations at an autosomal locus in human cells. Most involved large scale loss of genetic information, either through deletion or by recombination.

  8. Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture.

    Science.gov (United States)

    Steuernagel, Burkhard; Periyannan, Sambasivam K; Hernández-Pinzón, Inmaculada; Witek, Kamil; Rouse, Matthew N; Yu, Guotai; Hatta, Asyraf; Ayliffe, Mick; Bariana, Harbans; Jones, Jonathan D G; Lagudah, Evans S; Wulff, Brande B H

    2016-06-01

    Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5-15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution. If several cloned R genes were available, it would be possible to pyramid R genes in a crop, which might provide more durable resistance. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize.

  9. Strain improvement of Trametes hirsuta by physical and chemical mutagenesis for better laccases production.

    Science.gov (United States)

    Khanam, Rasheeda; Prasuna, R Gyana

    2013-07-01

    The Laccases production efficiency was investigated by the wild fungal strains Trametes hirsuta by treating it with physical mutagen [ultraviolet radiation (UV) and X-rays] and chemical mutagens [Ethidium bromide, Colchicine and Hydrogen peroxide]. The present work aimed to apply mutagenesis for enhancement of the enzyme production. Effective changes were observed in the efficiency of enzyme production when treated with physical and chemical mutagens. The effect of X-rays showed a decrease in production with increasing exposure in T. hirsuta (Max. at 2 sec.). UV irradiation influenced the enzyme production with higher exposure time (8 minutes) but the maximum dosage led to inhibition in fungal growth and low enzyme production. Among the three chemical mutagens used, hydrogen peroxide was found to be having lethal effects to the fungi but a minimum concentration (2 μg/mL) was positively effecting enzyme production. Colchicine showed increase in enzyme production with increasing concentrations (Max. at 9 μg) and with Ethidium bromide, maximum enzyme production was observed at concentration of 7 μg/ mL. The study on morphological differences in wild and mutant shows that there was an improvement in strains of the white rot fungi.

  10. ADA1 and NET1 Genes of Yeast Mediate Both Chromosome Maintenance and Mitochondrial $\\rho^{-}$ Mutagenesis

    CERN Document Server

    Koltovaya, N A; Tchekhouta, I A; Devin, A B

    2002-01-01

    An increase in the mitochondrial (mt) rho^- mutagenesis is a well-known respose of yeast cells to mutations in the numerous nuclear genes as well as to various kinds of stress. Notwithstanding the extensive studies during several decades the biological significance of this response is not yet fully understood. The genetic approach to solution of this subject includes the study of genes that are required for the high incidence of spontaneous rho^- mutants. Previously we found that mutations in certain nuclear genes including CDC28, the central cell-cycle regulation gene, may decrease the spontaneous rho^- mutability and simultaneously affect maintenance of the yeast chromosomes and plasmids. The present work provides data on identification of two more genes, resembling CDC28 in this respect. These genes NET1 and ADA1 mediate important regulatory protein-protein interactions in the yeast cell. The effects of net1 and ada1 mutations on the maintenance of yeast mt genome, chromosomes and plasmids as well as on ce...

  11. Towards Understanding the Catalytic Mechanism of Human Paraoxonase 1: Experimental and In Silico Mutagenesis Studies.

    Science.gov (United States)

    Tripathy, Rajan K; Aggarwal, Geetika; Bajaj, Priyanka; Kathuria, Deepika; Bharatam, Prasad V; Pande, Abhay H

    2017-08-01

    Human paraoxonase 1 (h-PON1) is a ~45-kDa serum enzyme that can hydrolyze a variety of substrates, including organophosphate (OP) compounds. It is a potential candidate for the development of antidote against OP poisoning in humans. However, insufficient OP-hydrolyzing activity of native enzyme affirms the urgent need to develop improved variant(s) having enhanced OP-hydrolyzing activity. The crystal structure of h-PON1 remains unsolved, and the molecular details of how the enzyme catalyses hydrolysis of different types of substrates are also not clear. Understanding the molecular details of the catalytic mechanism of h-PON1 is essential to engineer better variant(s) of enzyme. In this study, we have used a random mutagenesis approach to increase the OP-hydrolyzing activity of recombinant h-PON1. The mutants not only showed a 10-340-fold increased OP-hydrolyzing activity against different OP substrates but also exhibited differential lactonase and arylesterase activities. In order to investigate the mechanistic details of the effect of observed mutations on the hydrolytic activities of enzyme, molecular docking studies were performed with selected mutants. The results suggested that the observed mutations permit differential binding of substrate/inhibitor into the enzyme's active site. This may explain differential hydrolytic activities of the enzyme towards different substrates.

  12. Agrobacterium-mediated insertional mutagenesis in the mycorrhizal fungus Laccaria bicolor.

    Science.gov (United States)

    Stephan, B I; Alvarez Crespo, M C; Kemppainen, M J; Pardo, A G

    2017-05-01

    Agrobacterium-mediated gene transfer (AMT) is extensively employed as a tool in fungal functional genomics and accordingly, in previous studies we used AMT on a dikaryotic strain of the ectomycorrhizal basidiomycete Laccaria bicolor. The interest in this fungus derives from its capacity to establish a symbiosis with tree roots, thereby playing a major role in nutrient cycling of forest ecosystems. The ectomycorrhizal symbiosis is a highly complex interaction involving many genes from both partners. To advance in the functional characterization of fungal genes, AMT was used on a monokaryotic L. bicolor. A collection of over 1200 transgenic strains was produced, of which 200 randomly selected strains were analyzed for their genomic T-DNA insertion patterns. By means of insertional mutagenesis, a number of transgenic strains were obtained displaying differential growth features. Moreover, mating with a compatible strain resulted in dikaryons that retained altered phenotypic features of the transgenic monokaryon. The analysis of the T-DNA integration pattern revealed mostly similar results to those reported in earlier studies, confirming the usefulness of AMT on different genetic backgrounds of L. bicolor. Taken together, our studies display the great versatility and potentiality of AMT as a tool for the genetic characterization of L. bicolor.

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

    Science.gov (United States)

    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. Copyright © 2013. Published by Elsevier Ltd.

  14. Molecular properties and enhancement of thermostability by random mutagenesis of glutamate dehydrogenase from Bacillus subtilis.

    Science.gov (United States)

    Khan, Md Iqbal Hassan; Ito, Kousuke; Kim, Hyeung; Ashida, Hiroyuki; Ishikawa, Takahiro; Shibata, Hitoshi; Sawa, Yoshihiro

    2005-10-01

    The rocG gene encoding glutamate dehydrogenase from Bacillus subtilis (Bs-GluDH) was cloned, and expressed at considerable magnitude in Escherichia coli. The recombinant Bs-GluDH was purified to homogeneity and has been determined to have a hexameric structure (M(r) 270 kDa) with strict specificity for 2-oxoglutarate and L-glutamate, requiring NADH and NAD+ as cofactors respectively. The enzyme showed low thermostability with T(m) = 41 degrees C due to dissociation of the hexamer. To improve the thermostability of this enzyme, we performed error-prone PCR, introducing random mutagenesis on cloned GluDH. Two single mutant enzymes, Q144R and E27F, were isolated from the final mutant library. Their T(m) values were 61 degrees C and 49 degrees C respectively. Furthermore, Q144R had a remarkably high k(cat) value (435 s(-1)) for amination reaction at 37 degrees C, 1.3 times higher than that of the wild-type. Thus, Q144R can be used as a template gene to modify the substrate specificity of Bs-GluDH for industrial use.

  15. groE mutants of Escherichia coli are defective in umuDC-dependent UV mutagenesis

    International Nuclear Information System (INIS)

    Donnelly, C.E.; Walker, G.C.

    1989-01-01

    Overexpression of the SOS-inducible umuDC operon of Escherichia coli results in the inability of these cells to grow at 30 degrees C. Mutations in several heat shock genes suppress this cold sensitivity. Suppression of umuD+C+-dependent cold sensitivity appears to occur by two different mechanisms. We show that mutations in lon and dnaK heat shock genes suppress cold sensitivity in a lexA-dependent manner. In contrast, mutations in groES, groEL, and rpoH heat shock genes suppress cold sensitivity regardless of the transcriptional regulation of the umuDC genes. We have also found that mutations in groES and groEL genes are defective in umuDC-dependent UV mutagenesis. This defect can be suppressed by increased expression of the umuDC operon. The mechanism by which groE mutations affect umuDC gene product function may be related to the stability of the UmuC protein, since the half-life of this protein is shortened because of mutations at the groE locus

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

    Science.gov (United States)

    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 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.

  17. Random mutagenesis identifies factors involved in formate-dependent growth of the methanogenic archaeon Methanococcus maripaludis.

    Science.gov (United States)

    Sattler, Christian; Wolf, Sandro; Fersch, Julia; Goetz, Stefan; Rother, Michael

    2013-09-01

    Methane is a key intermediate in the carbon cycle and biologically produced by methanogenic archaea. Most methanogens are able to conserve energy by reducing CO2 to methane using molecular hydrogen as electron donor (hydrogenotrophic methanogenesis), but several hydrogenotrophic methanogens can also use formate as electron donor for methanogenesis. Formate dehydrogenase (Fdh) oxidizes formate to CO2 and is involved in funneling reducing equivalents into the methanogenic pathway, but details on other factors relevant for formate-dependent physiology of methanogens are not available. To learn more about the factors involved in formate-dependent growth of Methanococcus maripaludis strain JJ, we used a recently developed system for random in vitro mutagenesis, which is based on a modified insect transposable element to create 2,865 chromosomal transposon mutants and screened them for impaired growth on formate. Of 12 M. maripaludis transposon-induced mutants exhibiting this phenotype, the transposon insertion sites in the chromosome were mapped. Among the genes, apparently affecting formate-dependent growth were those encoding archaeal transcription factor S, a regulator of ion transport, and carbon monoxide dehydrogenase/acetyl-CoA synthase. Interestingly, in seven of the mutants, transposons were localized in a 10.2 kb region where Fdh1, one of two Fdh isoforms in the organism, is encoded. Two transcription start sites within the 10.2 kb region could be mapped, and quantification of transcripts revealed that transposon insertion in this region diminished fdhA1 expression due to polar effects.

  18. Mutagenesis of the repeat regions of herpesviruses cloned as bacterial artificial chromosomes.

    Science.gov (United States)

    Zhao, Yuguang; Nair, Venugopal

    2010-01-01

    Cloning of infectious and pathogenic herpesvirus genomes in a bacterial artificial chromosome (BAC) vector greatly facilitates genetic manipulation of their genomes. BAC-based mutagenesis strategies of viruses can advance our understanding of the viral gene functions and determinants of pathogenicity, and can ultimately help to develop molecularly defined improved vaccines against virus diseases. Unlike the virus stocks, where continuous passage in tissue culture can lead to phenotypic alterations such as loss of virulence or immunogenicity, viral genomes can be stably maintained with high fidelity as BAC clones in bacteria. Thanks to the "RecA" or the inducible phage "lambda Red" homologous recombination systems and a variety of positive and negative selection strategies, viral genomes cloned as BAC can be efficiently manipulated in E. coli. All the manipulations, including DNA fragment deletion or insertion, point mutations, or even multiple modifications in repeat regions can be carried out accurately in E. coli, and the mutated DNA can be used directly to reconstitute mutant viruses in transfected host cells. Furthermore, using self-excision strategies, the non-viral bacterial replicon sequence can be excised automatically during virus reconstitution, thus generating recombinant viruses virtually identical to the wild-type parent viruses. Here, we describe the various technologies of manipulating the infectious BAC clones of a group E herpesvirus as an example through a combination of different approaches.

  19. Quantitative mutagenesis by chemicals and by radiations: prerequisites for the establishment of rad-equivalences

    International Nuclear Information System (INIS)

    Latarjet, R.

    1977-01-01

    The lesions produced in the genetic material by chemical mutagens, on the one hand, and radiations, on the other, are very similar. In both cases, they are either lesions in DNA or changes in the bonds between this DNA and the proteins which surround it. The lesions are sufficiently similar to elicit, in both cases, the activity of the same repair systems. The similarity between chemical and radiation induced mutagenesis can be demonstrated by checking that a strain which is hyper-sensitive to radiation because it lacks some repair system, is also hyper-sensitive to most chemical mutagens. These similarities between the lesions suggest that one can establish an equivalence between the 'dose' of a chemical and a dose of radiation, on the basis of the effects produced on some biological systems of reference. Once such equivalence has been established, one could extrapolate the rules of radiation protection to protection against that chemical. Is this principle applicable, and under which conditions. What prerequisites must be fulfilled. The goal of this paper is to answer these questions

  20. Mutagenesis of mNeptune Red-Shifts Emission Spectrum to 681-685 nm.

    Science.gov (United States)

    Li, ZhaoYang; Zhang, ZhiPing; Bi, LiJun; Cui, ZongQiang; Deng, JiaoYu; Wang, DianBing; Zhang, Xian-En

    2016-01-01

    GFP-like fluorescent proteins with diverse emission wavelengths have been developed through mutagenesis, offering many possible choices in cellular and tissue imaging, such as multi-targets imaging, deep tissue imaging that require longer emission wavelength. Here, we utilized a combined approach of random mutation and structure-based rational design to develop new NIR fluorescent proteins on the basis of a far-red fluorescent protein, mNeptune (Ex/Em: 600/650 nm). We created a number of new monomeric NIR fluorescent proteins with the emission range of 681-685 nm, which exhibit the largest Stocks shifts (77-80 nm) compared to other fluorescent proteins. Among them, mNeptune681 and mNeptune684 exhibit more than 30 nm redshift in emission relative to mNeptune, owing to the major role of the extensive hydrogen-bond network around the chromophore and contributions of individual mutations to the observed redshift. Furthermore, the two variants still maintain monomeric state in solution, which is a trait crucial for their use as protein tags. In conclusion, our results suggest that there is untapped potential for developing fluorescent proteins with desired properties.

  1. Radiation-induced mutagenesis of antifungal metabolite producing bacillus sp. HKA-17

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young Keun; Senthilkumar, M. [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2009-09-15

    Bacillus sp. Strain HKA-17, isolated from the surface sterilized root nodule of Glycine max, inhibited several fungal plant pathogens. It produced a diffusible extracellular antifungal metabolite that was extracted with n-butanol. The crude extract was purified through Superdex{sup TM} 75 10/300 GL FPLC column. FT-IR spectrum of the FPLC purified-antifungal metabolite confirmed the presence of peptide and glycosidic bonds in its structure. Gamma induced mutagenesis of HKA-17 was carried out at an LD{sub 99} dose (8.46 kGy) to generate a mutant library. By screening the mutant library through a duel plate assay with Alternaria alternata, we selected one mutant with enhanced biocontrol activity (HKA-17e1) and two defective mutants (HKA-17d1 and HKA-17d2). Overproducing mutant recorded the largest inhibition zone (16.25 {+-} 0.86 mm) compared to any other mutant clone as well as wild type, and could be used as a potential biocontrol agent for plant disease suppression. The effect of HKA-17 antifungal metabolite on hyphal morphology was clearly demonstrated through scanning electron microscopy. The crude extract of defective mutant HKA-17 d1 did not induce any changes in hyphal morphology of A. alternata. However, antifungal metabolites of HKA-17 induced abnormal hyphal structures such as hyphal shrivelling, the bulging and swelling of intercalary cells, fragmentation, and cell lysis.

  2. ADA1 and NET1 genes of yeast mediate both chromosome maintenance and mitochondrial rho- mutagenesis

    International Nuclear Information System (INIS)

    Koltovaya, N.A.; Gerasimova, A.S.; Chekhuta, I.A.; Devin, A.B.

    2002-01-01

    An increase in the mitochondrial (mt) rho - mutagenesis is a well-known response of yeast cells to mutations in the numerous nuclear genes as well as to various kinds of stress. Notwithstanding the extensive studies during several decades the biological significance of this response is not yet fully understood. The genetic approach to solution of this subject includes the study of genes that are required for the high incidence of spontaneous rho - mutants. Previously we found that mutations in certain nuclear genes including CDC28, the central cell-cycle regulation gene, may decrease the spontaneous rho - mutability and simultaneously affect maintenance of the yeast chromosomes and plasmids. The present work provides data on identification of two more genes, resembling CDC28 in this respect. These genes NET1 and ADA1 mediate important regulatory protein-protein interactions in the yeast cell. The effects of net1 and ada1 mutations on the maintenance of yeast mt genome, chromosomes and plasmids as well on cell sensitivity to ionizing radiation are also described. (author)

  3. Mutagenesis of Xanthomonas campestris and selection of strains with enhanced Xanthan production

    International Nuclear Information System (INIS)

    Kamal, F.; Mehrgan, H.; Mazaheri, M.; Mortazavi, A. R.

    2003-01-01

    Xanthan gum is microbial polysaccharide of great commercial importance as it has been unusual rheological properties in solution and consequent range of applications. In this study, a series of mutants were isolated from Xanthomonas PTSS 1473 by ethyl methanesulfonate mutagenesis. The polysaccharide yield of one mutant, XC1473E 2 , was 30% better than that of the parent strain. It also showed higher xanthan formation of glucose consumption rates compared to the parent strain. xanthan produced by the mutant and enhanced viscosity, higher pseudo plasticity and larger molecular weight. Since mutant XC1473E 2 appeared white on agar plates, it underwent pigment extraction with methanol. Contrary to the parent strain, the mutant showed no absorption at 443 nm, i.e. the wavelength related to yellow pigment. This finding suggested that yellow pigmentation and normal xanthan biosynthesis are not necessarily concurrent. In general, mutant ZC1473e 2 seems to be a strain with interesting characteristics for use in commercial production of Xanthan

  4. Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations.

    Science.gov (United States)

    Maglennon, Gareth A; Cook, Beth S; Deeney, Alannah S; Bossé, Janine T; Peters, Sarah E; Langford, Paul R; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N

    2013-12-21

    Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.

  5. Enhancement of Biomass and Lipid Productivities of Water Surface-Floating Microalgae by Chemical Mutagenesis.

    Science.gov (United States)

    Nojima, Daisuke; Ishizuka, Yuki; Muto, Masaki; Ujiro, Asuka; Kodama, Fumito; Yoshino, Tomoko; Maeda, Yoshiaki; Matsunaga, Tadashi; Tanaka, Tsuyoshi

    2017-05-27

    Water surface-floating microalgae have great potential for biofuel applications due to the ease of the harvesting process, which is one of the most problematic steps in conventional microalgal biofuel production. We have collected promising water surface-floating microalgae and characterized their capacity for biomass and lipid production. In this study, we performed chemical mutagenesis of two water surface-floating microalgae to elevate productivity. Floating microalgal strains AVFF007 and FFG039 (tentatively identified as Botryosphaerella sp. and Chlorococcum sp., respectively) were exposed to ethyl methane sulfonate (EMS) or 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), and pale green mutants (PMs) were obtained. The most promising FFG039 PM formed robust biofilms on the surface of the culture medium, similar to those formed by wild type strains, and it exhibited 1.7-fold and 1.9-fold higher biomass and lipid productivities than those of the wild type. This study indicates that the chemical mutation strategy improves the lipid productivity of water surface-floating microalgae without inhibiting biofilm formation and floating ability.

  6. Molecular actions of Escherichia coli MutT for control of spontaneous mutagenesis.

    Science.gov (United States)

    Setoyama, Daiki; Ito, Riyoko; Takagi, Yasumitsu; Sekiguchi, Mutsuo

    2011-02-10

    MutT protein of Escherichia coli hydrolyzes oxidized guanine nucleotides, 8-oxo-dGTP and 8-oxoGTP, to the corresponding monophosphates, thereby preventing misincorporation of 8-oxoguanine into DNA and RNA, respectively. Although the biological significance of the MutT has been established, how MutT protein actually works in vivo remains to be elucidated. The current study shows the molecular behavior of the MutT protein in vivo and in vitro with special reference to control of spontaneous mutagenesis. A single E. coli cell carries about 70-75 molecules of the MutT protein and that this number does not change even when the cells were cultured in anaerobic and hyper-oxidative conditions. Conditional gene silencing analyses revealed that about a half number of MutT molecules are needed for keeping the spontaneous mutation frequency at the normal level. The MutT functions are not needed under anaerobic condition, yet the level of the MutT protein in cell is kept constant, probably for preparing for sudden changes of oxygen pressure. There is a possibility that MutT functions in close association with other proteins, and evidence is presented that MutT protein can interact with some proteins in vivo. Copyright © 2010 Elsevier B.V. All rights reserved.

  7. FoxP3 scanning mutagenesis reveals functional variegation and mild mutations with atypical autoimmune phenotypes.

    Science.gov (United States)

    Kwon, Ho-Keun; Chen, Hui-Min; Mathis, Diane; Benoist, Christophe

    2018-01-09

    FoxP3 + regulatory T cells (Tregs) are a central element of immunological tolerance. FoxP3 is the key determining transcription factor of the Treg lineage, interacting with numerous cofactors and transcriptional targets to determine the many facets of Treg function. Its absence leads to devastating lymphoproliferation and autoimmunity in scurfy mutant mice and immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) patients. To finely map transcriptionally active regions of the protein, with respect to disease-causing variation, we performed a systematic alanine-scan mutagenesis of FoxP3, assessing mutational impacts on DNA binding and transcriptional activation or repression. The mutations affected transcriptional activation and repression in a variegated manner involving multiple regions of the protein and varying between different transcriptional targets of FoxP3. There appeared to be different modalities for target genes related to classic immunosuppressive function vs. those related to atypical or tissue-Treg functions. Relevance to in vivo Treg biology was established by introducing some of the subtle Foxp3 mutations into the mouse germline by CRISPR-based genome editing. The resulting mice showed Treg populations in normal numbers and exhibited no overt autoimmune manifestations. However, Treg functional defects were revealed upon competition or by system stress, manifest as a strikingly heightened susceptibility to provoked colitis, and conversely by greater resistance to tumors. These observations suggest that some of the missense mutations that segregate in human populations, but do not induce IPEX manifestations, may have unappreciated consequences in other diseases.

  8. Improving the thermostability of alpha-amylase by combinatorial coevolving-site saturation mutagenesis

    Directory of Open Access Journals (Sweden)

    Wang Chenghua

    2012-10-01

    Full Text Available Abstract Background The generation of focused mutant libraries at hotspot residues is an important strategy in directed protein evolution. Existing methods, such as combinatorial active site testing and residual coupling analysis, depend primarily on the evolutionary conserved information to find the hotspot residues. Hardly any attention has been paid to another important functional and structural determinants, the functionally correlated variation information--coevolution. Results In this paper, we suggest a new method, named combinatorial coevolving-site saturation mutagenesis (CCSM, in which the functionally correlated variation sites of proteins are chosen as the hotspot sites to construct focused mutant libraries. The CCSM approach was used to improve the thermal stability of α-amylase from Bacillus subtilis CN7 (Amy7C. The results indicate that the CCSM can identify novel beneficial mutation sites, and enhance the thermal stability of wild-type Amy7C by 8°C (T5030, which could not be achieved with the ordinarily rational introduction of single or a double point mutation. Conclusions Our method is able to produce more thermostable mutant α-amylases with novel beneficial mutations at new sites. It is also verified that the coevolving sites can be used as the hotspots to construct focused mutant libraries in protein engineering. This study throws new light on the active researches of the molecular coevolution.

  9. Rose (Rosa hybrida L.) tissue culture mutagenesis for new mutants generation

    International Nuclear Information System (INIS)

    Salahbiah Abdul Majid; Rusli Ibrahim

    2004-01-01

    Tissue culture technique can be used to obtain complete regeneration of plant cells from shoots, rots, flowers, axillary buds and other parts of the plant. In this study, axillary buds from stem cuttings of Cutting Red, Christine Dior and Mini Rose varieties were used as the stating explants. Murashige and Skoog (1962) media supplemented with 6-Benzylaminopurine (BAP, at 4.44 - 8.88μM/l), Napthaleneacetic acid (NAA at 0.54μM/l),, nad 3% sucrose were used for plantlet initiation and regeneration. Cultured axillary buds were exposed to gamma ray (0.250 Gy/s) at 0, 15, 25, 35, 45, 55, 65 and 75 Gy for radiosensitivity test. From the dose respond curve, LD 5 0 the value for cutting red variety was 25 Gy, Christion Dior 30 Gy and Mini Rose 38 Gy, yet 22% of Mini Rose samples survived at 65 Gy and another 10% at 70 Gy. Screening of M3 plants of irradiated cultured shoots, 2 colour variations were obtained at 40 Gy for Cutting Red variety, while 3 colour variations for Mini Rose at 20 Gy. When 6 varieties of Fragrance Rose were irradiated at 40 Gy, 1 colour variation was obtained from 99 screened plants. This study suggests that the dose range of 20 to 45 can be considered for rose mutagenesis study to produce mutants. (Author)

  10. The fitness landscape of HIV-1 gag: advanced modeling approaches and validation of model predictions by in vitro testing.

    Directory of Open Access Journals (Sweden)

    Jaclyn K Mann

    2014-08-01

    Full Text Available Viral immune evasion by sequence variation is a major hindrance to HIV-1 vaccine design. To address this challenge, our group has developed a computational model, rooted in physics, that aims to predict the fitness landscape of HIV-1 proteins in order to design vaccine immunogens that lead to impaired viral fitness, thus blocking viable escape routes. Here, we advance the computational models to address previous limitations, and directly test model predictions against in vitro fitness measurements of HIV-1 strains containing multiple Gag mutations. We incorporated regularization into the model fitting procedure to address finite sampling. Further, we developed a model that accounts for the specific identity of mutant amino acids (Potts model, generalizing our previous approach (Ising model that is unable to distinguish between different mutant amino acids. Gag mutation combinations (17 pairs, 1 triple and 25 single mutations within these predicted to be either harmful to HIV-1 viability or fitness-neutral were introduced into HIV-1 NL4-3 by site-directed mutagenesis and replication capacities of these mutants were assayed in vitro. The predicted and measured fitness of the corresponding mutants for the original Ising model (r = -0.74, p = 3.6×10-6 are strongly correlated, and this was further strengthened in the regularized Ising model (r = -0.83, p = 3.7×10-12. Performance of the Potts model (r = -0.73, p = 9.7×10-9 was similar to that of the Ising model, indicating that the binary approximation is sufficient for capturing fitness effects of common mutants at sites of low amino acid diversity. However, we show that the Potts model is expected to improve predictive power for more variable proteins. Overall, our results support the ability of the computational models to robustly predict the relative fitness of mutant viral strains, and indicate the potential value of this approach for understanding viral immune evasion

  11. The fitness landscape of HIV-1 gag: advanced modeling approaches and validation of model predictions by in vitro testing.

    Science.gov (United States)

    Mann, Jaclyn K; Barton, John P; Ferguson, Andrew L; Omarjee, Saleha; Walker, Bruce D; Chakraborty, Arup; Ndung'u, Thumbi

    2014-08-01

    Viral immune evasion by sequence variation is a major hindrance to HIV-1 vaccine design. To address this challenge, our group has developed a computational model, rooted in physics, that aims to predict the fitness landscape of HIV-1 proteins in order to design vaccine immunogens that lead to impaired viral fitness, thus blocking viable escape routes. Here, we advance the computational models to address previous limitations, and directly test model predictions against in vitro fitness measurements of HIV-1 strains containing multiple Gag mutations. We incorporated regularization into the model fitting procedure to address finite sampling. Further, we developed a model that accounts for the specific identity of mutant amino acids (Potts model), generalizing our previous approach (Ising model) that is unable to distinguish between different mutant amino acids. Gag mutation combinations (17 pairs, 1 triple and 25 single mutations within these) predicted to be either harmful to HIV-1 viability or fitness-neutral were introduced into HIV-1 NL4-3 by site-directed mutagenesis and replication capacities of these mutants were assayed in vitro. The predicted and measured fitness of the corresponding mutants for the original Ising model (r = -0.74, p = 3.6×10-6) are strongly correlated, and this was further strengthened in the regularized Ising model (r = -0.83, p = 3.7×10-12). Performance of the Potts model (r = -0.73, p = 9.7×10-9) was similar to that of the Ising model, indicating that the binary approximation is sufficient for capturing fitness effects of common mutants at sites of low amino acid diversity. However, we show that the Potts model is expected to improve predictive power for more variable proteins. Overall, our results support the ability of the computational models to robustly predict the relative fitness of mutant viral strains, and indicate the potential value of this approach for understanding viral immune evasion, and

  12. Development of an Asymbiotic Reference Line for Pea cv. Bohatýr by De Novo Mutagenesis

    Czech Academy of Sciences Publication Activity Database

    Novák, Karel; Šlajs, Martin; Biedermannová, Eva; Vondrys, Josef

    2005-01-01

    Roč. 45, - (2005), s. 1837-1843 ISSN 0011-183X R&D Projects: GA ČR GA521/00/0937; GA ČR GA521/03/0192 Institutional research plan: CEZ:AV0Z50200510 Keywords : mutagenesis * bohatýr * nitrogen fixation Subject RIV: EE - Microbiology, Virology Impact factor: 0.925, year: 2005

  13. Optimization of lipid production by the oleaginous yeast Lipomyces starkeyi by random mutagenesis coupled to cerulenin screening

    OpenAIRE

    Tapia V, Eulalia; Anschau, Andr?ia; Coradini, Alessandro LV; T Franco, Telma; Deckmann, Ana Carolina

    2012-01-01

    In this work we performed assays for the genetic improvement of the oleaginous yeast Lipomyces starkeyi DSM 70296 focusing on its utilization for lipid biosynthesis from renewable sources. The genetic optimization was carried out by random mutagenesis by ultraviolet irradiation and mutant selection by cerulenin, a compound displaying inhibitory effects on lipid biosynthesis. Mutants demonstrating normal growth in presence of cerulenin were considered as good candidates for further studies. Us...

  14. Rapid Discovery of Continuous-Performance Compounds and Powernap Compounds Through Large-Scale Mutagenesis in Drosophila

    Science.gov (United States)

    2008-04-22

    REPORT Rapid Discovery of Continuous-Performance Compounds and Powernap Compounds Through Large-Scale Mutagenesis in Drosophila 14. ABSTRACT 16...sustained wakefulness” (R. Benca-PI, N. Rattenborg-CoPI). The goal of both projects was to find ways to postpone temporarily the need for sleep ...Research Triangle Park, NC 27709-2211 15. SUBJECT TERMS drosophila short sleeping phenotype Kv potassium channel migration Giulio Tononi, Ruth

  15. Combinatorial effect of mutagenesis and medium component optimization on Bacillus amyloliquefaciens antifungal activity and efficacy in eradicating Botrytis cinerea.

    Science.gov (United States)

    Masmoudi, Fatma; Ben Khedher, Saoussen; Kamoun, Amel; Zouari, Nabil; Tounsi, Slim; Trigui, Mohamed

    2017-04-01

    This work is directed towards Bacillus amyloliquefaciens strain BLB371 metabolite production for biocontrol of fungal phytopathogens. In order to maximise antifungal metabolite production by this strain, two approaches were combined: random mutagenesis and medium component optimization. After three rounds of mutagenesis, a hyper active mutant, named M3-7, was obtained. It produces 7 fold more antifungal metabolites (1800AU/mL) than the wild strain in MC medium. A hybrid design was applied to optimise a new medium to enhance antifungal metabolite production by M3-7. The new optimized medium (35g/L of peptone, 32.5g/L of sucrose, 10.5g/L of yeast extract, 2.4g/L of KH 2 PO 4 , 1.3g/L of MgSO 4 and 23mg/L of MnSO 4 ) achieved 1.62 fold enhancement in antifungal compound production (3000AU/mL) by this mutant, compared to that achieved in MC medium. Therefore, combinatory effect of these two approaches (mutagenesis and medium component optimization) allowed 12 fold improvement in antifungal activity (from 250UA/mL to 3000UA/mL). This improvement was confirmed against several phytopathogenic fungi with an increase of MIC and MFC over than 50%. More interestingly, a total eradication of gray mold was obtained on tomato fruits infected by Botrytis cinerea and treated by M3-7, compared to those treated by BLB371. From the practical point of view, combining random mutagenesis and medium optimization could be considered as an excellent tool for obtaining promising biological products useful against phytopathogenic fungi. Copyright © 2017 Elsevier GmbH. All rights reserved.

  16. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

    OpenAIRE

    Weir Jerry P; Schmeisser Falko

    2007-01-01

    Abstract Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. Whi...

  17. Rice improvement involving altered flower structure more suitable to cross-pollination, using in vitro culture in combination with mutagenesis

    International Nuclear Information System (INIS)

    Min, S.K.

    1998-01-01

    Anther and somatic tissue culture in combination with mutagenesis were carried out to evaluate the efficiency of different mutagenic treatments of various in vitro culture materials, and to obtain some promising variants for rice improvement. Results indicated that in japonica rice radiation treatment of dry seeds and young panicles influenced the percentage of green plantlets regeneration from anther culture. Both treatments increased significantly the percentage of regenerated green plant lets in comparison with the control

  18. Molecular modeling and docking study on dopamine D2-like and serotonin 5-HT2A receptors.

    Science.gov (United States)

    Duan, Xinli; Zhang, Min; Zhang, Xin; Wang, Fang; Lei, Ming

    2015-04-01

    Psychiatric disorders, such as schizophrenia, bipolar disorder and major depression, are paid more and more attention by human due to their upward tendency in modern society. D2-like and 5-HT2A receptors have been proposed as targets of antipsychotic drugs. Atypical antipsychotic drugs have been deemed to improve the treatment of positive, negative and extrapyramidal symptoms. Unfortunately, no experimental structures for these receptors are available except D3 receptor (D3R). Therefore, it is necessary to construct structures of D2-like and 5-HT2A receptors to investigate the interaction between these receptors and their antagonists. Accordingly, homology models of dopamine D2, D3, D4 and serotonin 5-HT2A receptors have been built on the high-resolution crystal structure of the β2-adrenergic receptor, and refined by molecular dynamics simulations. The backbone root-mean-square deviation (RMSD) of D3R model relative to crystal structure is 1.3Å, which proves the reliability of homology modeling. Docking studies reveal that the binding modes of four homology models and their antagonists are consistent with experimental site-directed mutagenesis data. The calculated pKi values agree well with the experimental pKi ones. Antagonists with linear structures such as butyrophenones and benzisoxazolyl piperidines are easily docked into D2-like and 5-HT2A receptors. Polycyclic aromatic compounds have weaker affinity with four receptors. Homology models of D2-like and 5-HT2A receptors will be helpful for predicting the affinity of novel ligands, and could be used as three-dimensional (3D) templates for antipsychotic virtual screening and further drug discovery. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Transcriptome analysis of Neisseria meningitidis in human whole blood and mutagenesis studies identify virulence factors involved in blood survival.

    Directory of Open Access Journals (Sweden)

    Hebert Echenique-Rivera

    2011-05-01

    Full Text Available During infection Neisseria meningitidis (Nm encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating

  20. Confirming therapeutic target of protopine using immobilized β2 -adrenoceptor coupled with site-directed molecular docking and the target-drug interaction by frontal analysis and injection amount-dependent method.

    Science.gov (United States)

    Liu, Guangxin; Wang, Pei; Li, Chan; Wang, Jing; Sun, Zhenyu; Zhao, Xinfeng; Zheng, Xiaohui

    2017-07-01

    Drug-protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β 2 -adrenoceptor (β 2 -AR) by linkage of the receptor on macroporous silica gel surface through N,N'-carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site-directed molecular docking. Subsequent application of immobilized β 2 -AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount-dependent method. The association constants of protopine to β 2 -AR by the 2 methods were (1.00 ± 0.06) × 10 5 M -1 and (1.52 ± 0.14) × 10 4 M -1 . The numbers of binding sites were (1.23 ± 0.07) × 10 -7 M and (9.09 ± 0.06) × 10 -7 M, respectively. These results indicated that β 2 -AR is the specific target for therapeutic action of protopine in vivo. The target-drug binding occurred on Ser 169 in crystal structure of the receptor. Compared with frontal analysis, injection amount-dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high-throughput drug-receptor interaction analysis. Copyright © 2017 John Wiley & Sons, Ltd.

  1. A streptomycin resistance marker in H. parasuis based on site-directed mutations in rpsL gene to perform unmarked in-frame mutations and to verify natural transformation

    Directory of Open Access Journals (Sweden)

    Ke Dai

    2018-01-01

    Full Text Available Haemophilus parasuis is a member of the family Pasteurellaceae and a major causative agent of Glässer’s disease. This bacterium is normally a benign swine commensal but may become a deadly pathogen upon penetration into multiple tissues, contributing to severe lesions in swine. We have established a successive natural transformation-based markerless mutation system in this species. However, the two-step mutation system requires screening of natural competent cells, and cannot delete genes which regulate natural competence per se. In this study, we successfully obtained streptomycin-resistant derivatives from H. parasuis wild type strain SC1401 by using ethyl methane sulfonate (EMS, CH3SO2OC2H5. Upon sequencing and site-directed mutations, we uncovered that the EMS-induced point mutation in rpsL at codon 43rd (AAA → AGA; K43R or at 88th (AAA → AGA; K88R confers a much higher streptomycin resistance than clinical isolates. We have applied the streptomycin resistance marker as a positive selection marker to perform homologous recombination through conjugation and successfully generated a double unmarked in-frame targeted mutant 1401D88△tfox△arcA. Combined with a natural transformation-based knockout system and this genetic technique, multiple deletion mutants or attenuated strains of H. parasuis can be easily constructed. Moreover, the mutant genetic marker rpsL and streptomycin resistant phenotypes can serve as an effective tool to select naturally competent strains, and to verify natural transformation quantitatively.

  2. A streptomycin resistance marker inH. parasuisbased on site-directed mutations inrpsLgene to perform unmarked in-frame mutations and to verify natural transformation.

    Science.gov (United States)

    Dai, Ke; Wen, Xintian; Chang, Yung-Fu; Cao, Sanjie; Zhao, Qin; Huang, Xiaobo; Wu, Rui; Huang, Yong; Yan, Qigui; Han, Xinfeng; Ma, Xiaoping; Wen, Yiping

    2018-01-01

    Haemophilus parasuis is a member of the family Pasteurellaceae and a major causative agent of Glässer's disease. This bacterium is normally a benign swine commensal but may become a deadly pathogen upon penetration into multiple tissues, contributing to severe lesions in swine. We have established a successive natural transformation-based markerless mutation system in this species. However, the two-step mutation system requires screening of natural competent cells, and cannot delete genes which regulate natural competence per se. In this study, we successfully obtained streptomycin-resistant derivatives from H. parasuis wild type strain SC1401 by using ethyl methane sulfonate (EMS, CH 3 SO 2 OC 2 H 5 ). Upon sequencing and site-directed mutations, we uncovered that the EMS-induced point mutation in rpsL at codon 43rd (AAA → AGA; K43R) or at 88th (AAA → AGA; K88R) confers a much higher streptomycin resistance than clinical isolates. We have applied the streptomycin resistance marker as a positive selection marker to perform homologous recombination through conjugation and successfully generated a double unmarked in-frame targeted mutant 1401D88△ tfox △ arcA . Combined with a natural transformation-based knockout system and this genetic technique, multiple deletion mutants or attenuated strains of H. parasuis can be easily constructed. Moreover, the mutant genetic marker rpsL and streptomycin resistant phenotypes can serve as an effective tool to select naturally competent strains, and to verify natural transformation quantitatively.

  3. Structure of Streptococcus agalactiae tip pilin GBS104: a model for GBS pili assembly and host interactions

    Energy Technology Data Exchange (ETDEWEB)

    Krishnan, Vengadesan [UNESCO Regional Centre for Biotechnology (RCB), Gurgaon 122 016, Haryana (India); Dwivedi, Prabhat [University of Texas Health Science Center, Houston, TX 77030 (United States); Kim, Brandon J. [San Diego State University, 5500 Campanile Drive, San Diego, CA 92182 (United States); Samal, Alexandra; Macon, Kevin [University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Ma, Xin; Mishra, Arunima [University of Texas Health Science Center, Houston, TX 77030 (United States); Doran, Kelly S. [San Diego State University, 5500 Campanile Drive, San Diego, CA 92182 (United States); Ton-That, Hung [University of Texas Health Science Center, Houston, TX 77030 (United States); Narayana, Sthanam V. L., E-mail: narayana@uab.edu [University of Alabama at Birmingham, Birmingham, AL 35294 (United States); UNESCO Regional Centre for Biotechnology (RCB), Gurgaon 122 016, Haryana (India)

    2013-06-01

    The crystal structure of a 75 kDa central fragment of GBS104, a tip pilin from the 2063V/R strain of Streptococcus agalactiae (group B streptococcus; GBS), is reported. The crystal structure of a 75 kDa central fragment of GBS104, a tip pilin from the 2063V/R strain of Streptococcus agalactiae (group B streptococcus; GBS), is reported. In addition, a homology model of the remaining two domains of GBS104 was built and a model of full-length GBS104 was generated by combining the homology model (the N1 and N4 domains) and the crystal structure of the 75 kDa fragment (the N2 and N3 domains). This rod-shaped GBS104 model is constructed of three IgG-like domains (the N1, N2 and N4 domains) and one vWFA-like domain (the N3 domain). The N1 and N2 domains of GBS104 are assembled with distinct and remote segments contributed by the N- and C-termini. The metal-binding site in the N3 domain of GBS104 is in the closed/low-affinity conformation. Interestingly, this domain hosts two long arms that project away from the metal-binding site. Using site-directed mutagenesis, two cysteine residues that lock the N3 domain of GBS104 into the open/high-affinity conformation were introduced. Both wild-type and disulfide-locked recombinant proteins were tested for binding to extracellular matrix proteins such as collagen, fibronectin, fibrinogen and laminin, and an increase in fibronectin binding affinity was identified for the disulfide-locked N3 domain, suggesting that induced conformational changes may play a possible role in receptor binding.

  4. Meeting Report. Assessing Human Germ-Cell Mutagenesis in thePost-Genome Era: A Celebration of the Legacy of William Lawson (Bill)Russell

    Energy Technology Data Exchange (ETDEWEB)

    Wyrobek, Andrew J.; Mulvihill, John J.; Wassom, John S.; Malling,Heinrich V.; Shelby, Michael D.; Lewis, Susan E.; Witt, Kristine L.; Preston, R. Julian; Perreault-Darney, Sally; Allen, James W.; DeMarini,David M.; Woychik, Richard P.; Bishop Jack B; Workshop Presenters

    2006-04-18

    Although numerous germ-cell mutagens have been identified inanimal model systems, to date, no human germ-cell mutagens have beenconfirmed. Because the genomic integrity of our germ cells is essentialfor the continuation of the human species, a resolution of this enduringconundrum is needed. To facilitate such a resolution, we organized aworkshop at The Jackson Laboratory in Bar Harbor, Maine on September28-30, 2004. This interactive workshop brought together scientists from awide range of disciplines to assess the applicability of emergingmolecular methods for genomic analysis to the field of human germ-cellmutagenesis. Participants recommended that focused, coordinated humangerm-cell mutation studies be conducted in relation to important societalexposures. Because cancer survivors represent a unique cohort withwell-defined exposures, there was a consensus that studies should bedesigned to assess the mutational impact on children born to parents whohad received certain types of mutagenic cancer chemotherapy prior toconceiving their children. Within this high-risk cohort, parents andchildren could be evaluated for inherited changes in (a) gene sequencesand chromosomal structure, (b) repeat sequences and minisatelliteregions, and (c) global gene expression and chromatin. Participants alsorecommended studies to examine trans-generational effects in humansinvolving mechanisms such as changes in imprinting and methylationpatterns, expansion of nucleotide repeats, or induction of mitochondrialDNA mutations. Workshop participants advocated establishment of abio-bank of human tissue samples that could be used to conduct amultiple-endpoint, comprehensive, and collaborative effort to detectexposure-induced heritable alterations in the human genome. Appropriateanimal models of human germ-cell mutagenes is should be used in parallelwith human studies to provide insights into the mechanisms of mammaliangerm-cell mutagenesis. Finally, participants recommended that

  5. In vitro mutagenesis for development of novel variants in Rose (Rosa X Hybrida)

    International Nuclear Information System (INIS)

    Namita, B.; Raju, D.V.S.; Prasad, K.V.; Singh, Kanwar P.

    2014-01-01

    Rose (Rosa x hybrida) is the top ranking cut flower in international market due to its attractive flowers with longer stem and fragrance. Modern roses are the result of hybridization, selection and spontaneous mutations. In floriculture industry, the demand of the novel varieties of rose is increasing day by day due to change in trends and taste. Mutation breeding is an already established method for improvement of flower crops. It offers several advantages over conventional methods for the improvement of one or more traits within a short span of time. Induced mutation is one of the potent methods to induce variability in existing rose genotypes. Mutation breeding combined with tissue culture i.e. in vitro mutagenesis has proven more effective in rose due to controlled environment that provides ideal conditions for survival of mutated tissues or cells. Keeping these aspects in view, the present investigation was carried out to induce variability in rose cv. Grand Gala under in vitro conditions using gamma (γ) rays. Single node cuttings were excised from the field grown plants and were irradiated with different doses of γ rays (0,10, 20, 30, 40, 50, 60, 70 and 80 Gy) using a 60 Co source. The γ-irradiated explants were then pretreated, surface sterilized and cultured aseptically on Murashige and Skoog basal medium supplemented with 2.5 mg 5-benzylaminopurine (BAP) plus 5.0 mg kinetin plus 0.1 mg a-naphthaleneacetic acid (NAA) plus 0.5 mg gibberellic acid (GA 3 ) plus 40 mg adenine sulphate plus 0.8% (w/v) agar-agar to induce sprouting and shoot proliferation. Explants treated at higher dose of γ-rays (70 and 80 Gy) showed deleterious effects of ionising radiation. The 30 Gy treatment was found to be the LD 50 dose. It was observed that few explants treated with γ-rays sprouted, showed slow growth and failed to survive after the first sub culture. The explants irradiated with 50 Gy γ-rays exhibited minimum explant survival, bud sprouting, number of shoots

  6. Enhancement of Lutein Production in Chlorella sorokiniana (Chorophyta) by Improvement of Culture Conditions and Random Mutagenesis

    Science.gov (United States)

    Cordero, Baldo F.; Obraztsova, Irina; Couso, Inmaculada; Leon, Rosa; Vargas, Maria Angeles; Rodriguez, Herminia

    2011-01-01

    Chlorella sorokiniana has been selected for lutein production, after a screening of thirteen species of microalgae, since it showed both a high content in this carotenoid and a high growth rate. The effects of several nutritional and environmental factors on cell growth and lutein accumulation have been studied. Maximal specific growth rate and lutein content were attained at 690 μmol photons m−2 s−1, 28 °C, 2 mM NaCl, 40 mM nitrate and under mixotrophic conditions. In general, optimal conditions for the growth of this strain also lead to maximal lutein productivity. High lutein yielding mutants of C. sorokiniana have been obtained by random mutagenesis, using N-methyl-N′-nitro-nitrosoguanidine (MNNG) as a mutagen and selecting mutants by their resistance to the inhibitors of the carotenogenic pathway nicotine and norflurazon. Among the mutants resistant to the herbicides, those exhibiting both high content in lutein and high growth rate were chosen. Several mutants exhibited higher contents in this carotenoid than the wild type, showing, in addition, either a similar or higher growth rate than the latter strain. The mutant MR-16 exhibited a 2.0-fold higher volumetric lutein content than that of the wild type, attaining values of 42.0 mg L−1 and mutants DMR-5 and DMR-8 attained a lutein cellular content of 7.0 mg g−1 dry weight. The high lutein yield exhibited by C. sorokiniana makes this microalga an excellent candidate for the production of this commercially interesting pigment. PMID:22131961

  7. Charged particle mutagenesis at low dose and fluence in mouse splenic T cells

    Energy Technology Data Exchange (ETDEWEB)

    Grygoryev, Dmytro [Oregon Institute of Occupational Health Sciences, Oregon Health & Science University, Portland, OR 97239 (United States); Gauny, Stacey [Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Lasarev, Michael; Ohlrich, Anna [Oregon Institute of Occupational Health Sciences, Oregon Health & Science University, Portland, OR 97239 (United States); Kronenberg, Amy [Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Turker, Mitchell S., E-mail: turkerm@ohsu.edu [Oregon Institute of Occupational Health Sciences, Oregon Health & Science University, Portland, OR 97239 (United States); Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR 97239 (United States)

    2016-06-15

    Highlights: • Densely ionizing forms of space radiation induce mutations in splenic T cells at low fluence. • Large interstitial deletions and discontinuous LOH patterns are radiation signature mutations. • Space radiation mutagenesis suggests a cancer risk from deep space travel. - Abstract: High-energy heavy charged particles (HZE ions) found in the deep space environment can significantly affect human health by inducing mutations and related cancers. To better understand the relation between HZE ion exposure and somatic mutation, we examined cell survival fraction, Aprt mutant frequencies, and the types of mutations detected for mouse splenic T cells exposed in vivo to graded doses of densely ionizing {sup 48}Ti ions (1 GeV/amu, LET = 107 keV/μm), {sup 56}Fe ions (1 GeV/amu, LET = 151 keV/μm) ions, or sparsely ionizing protons (1 GeV, LET = 0.24 keV/μm). The lowest doses for {sup 48}Ti and {sup 56}Fe ions were equivalent to a fluence of approximately 1 or 2 particle traversals per nucleus. In most cases, Aprt mutant frequencies in the irradiated mice were not significantly increased relative to the controls for any of the particles or doses tested at the pre-determined harvest time (3–5 months after irradiation). Despite the lack of increased Aprt mutant frequencies in the irradiated splenocytes, a molecular analysis centered on chromosome 8 revealed the induction of radiation signature mutations (large interstitial deletions and complex mutational patterns), with the highest levels of induction at 2 particles nucleus for the {sup 48}Ti and {sup 56}Fe ions. In total, the results show that densely ionizing HZE ions can induce characteristic mutations in splenic T cells at low fluence, and that at least a subset of radiation-induced mutant cells are stably retained despite the apparent lack of increased mutant frequencies at the time of harvest.

  8. The induced mutagenesis and the genetic progress in the work with sour cherry

    International Nuclear Information System (INIS)

    Zhukov, O.S.

    1990-01-01

    Full text: We used radiation breeding for improving sour cherry varieties since 1958. X- and gamma-rays and also other physical factors have been applied to cuttings, seeds, flower parts and whole plants as well as in-vitro cultures. Doses of 30-60 Gy appeared to be most effective for inducing mutation when cuttings were irradiated. The main number of mutations occurred in M 1 and M 2 . Mutations were divided into 5 main classes concerning morphological characters, tree growth, dates of fruit bearing, biochemical composition, system of propagation. As a result of x-irradiation of a sour cherry/bird cherry hybrid 'Padocerus', immune to Coccomyces hiemalis, a highly fertile mutant 'Padocerus M' has been obtained. A dominant gene has been identified, controlling resistance to Coccomyces hiemalis. By obtaining 'Padocerus M' the possibilities of increasing the genetic resources have been expanded. 'Almaz' is the monogenic donor of resistance to C. hiemalis. In literature there are indications of the possibility of obtaining apomictic forms by mutagenesis. As a result of irradiating 'Padocerus' plants in the gamma field during three years a mutant has been found in which the basic mass of seeds is formed as a result of apomixis-autonomous diplosporic parthenogenesis. Apomixis seems to be controlled by a small number of major and minor genes. The mutant is called 'Padocerus A' and is used in hybridisation with other sour cherry varieties; segregation for the apomictic type of propagation has been found. A population of Prunus fructicosa has been studied in the region of the so-called Tatar bank in the Tambov district. Large-fruited forms have been selected which may be the result of accumulating spontaneous mutations. Mutations of a different type have been obtained when using chemical mutagens or a laser beam. (author)

  9. Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci.

    Science.gov (United States)

    Westrick, Randal J; Tomberg, Kärt; Siebert, Amy E; Zhu, Guojing; Winn, Mary E; Dobies, Sarah L; Manning, Sara L; Brake, Marisa A; Cleuren, Audrey C; Hobbs, Linzi M; Mishack, Lena M; Johnston, Alexander J; Kotnik, Emilee; Siemieniak, David R; Xu, Jishu; Li, Jun Z; Saunders, Thomas L; Ginsburg, David

    2017-09-05

    Factor V Leiden ( F5 L ) is a common genetic risk factor for venous thromboembolism in humans. We conducted a sensitized N -ethyl- N -nitrosourea (ENU) mutagenesis screen for dominant thrombosuppressor genes based on perinatal lethal thrombosis in mice homozygous for F5 L ( F5 L/L ) and haploinsufficient for tissue factor pathway inhibitor ( Tfpi +/- ). F8 deficiency enhanced the survival of F5 L/L Tfpi +/- mice, demonstrating that F5 L/L Tfpi +/- lethality is genetically suppressible. ENU-mutagenized F5 L/L males and F5 L/+ Tfpi +/- females were crossed to generate 6,729 progeny, with 98 F5 L/L Tfpi +/- offspring surviving until weaning. Sixteen lines, referred to as "modifier of Factor 5 Leiden ( MF5L1-16 )," exhibited transmission of a putative thrombosuppressor to subsequent generations. Linkage analysis in MF5L6 identified a chromosome 3 locus containing the tissue factor gene ( F3 ). Although no ENU-induced F3 mutation was identified, haploinsufficiency for F3 ( F3 +/- ) suppressed F5 L/L Tfpi +/- lethality. Whole-exome sequencing in MF5L12 identified an Actr2 gene point mutation (p.R258G) as the sole candidate. Inheritance of this variant is associated with suppression of F5 L/L Tfpi +/- lethality ( P = 1.7 × 10 -6 ), suggesting that Actr2 p.R258G is thrombosuppressive. CRISPR/Cas9 experiments to generate an independent Actr2 knockin/knockout demonstrated that Actr2 haploinsufficiency is lethal, supporting a hypomorphic or gain-of-function mechanism of action for Actr2 p.R258G Our findings identify F8 and the Tfpi/F3 axis as key regulators in determining thrombosis balance in the setting of F5 L and also suggest a role for Actr2 in this process.

  10. A regulatory role for NBS1 in strand-specific mutagenesis during somatic hypermutation.

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    Likun Du

    2008-06-01

    Full Text Available Activation-induced cytidine deaminase (AID is believed to initiate somatic hypermutation (SHM by deamination of deoxycytidines to deoxyuridines within the immunoglobulin variable regions genes. The deaminated bases can subsequently be replicated over, processed by base excision repair or mismatch repair, leading to introduction of different types of point mutations (G/C transitions, G/C transversions and A/T mutations. It is evident that the base excision repair pathway is largely dependent on uracil-DNA glycosylase (UNG through its uracil excision activity. It is not known, however, which endonuclease acts in the step immediately downstream of UNG, i.e. that cleaves at the abasic sites generated by the latter. Two candidates have been proposed, an apurinic/apyrimidinic endonuclease (APE and the Mre11-Rad50-NBS1 complex. The latter is intriguing as this might explain how the mutagenic pathway is primed during SHM. We have investigated the latter possibility by studying the in vivo SHM pattern in B cells from ataxia-telangiectasia-like disorder (Mre11 deficient and Nijmegen breakage syndrome (NBS1 deficient patients. Our results show that, although the pattern of mutations in the variable heavy chain (V(H genes was altered in NBS1 deficient patients, with a significantly increased number of G (but not C transversions occurring in the SHM and/or AID targeting hotspots, the general pattern of mutations in the V(H genes in Mre11 deficient patients was only slightly altered, with an increased frequency of A to C transversions. The Mre11-Rad50-NBS1 complex is thus unlikely to be the major nuclease involved in cleavage of the abasic sites during SHM, whereas NBS1 might have a specific role in regulating the strand-biased repair during phase Ib mutagenesis.

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

    Science.gov (United States)

    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.

  12. WR-2721 protects against cytoxan-induced hprt mutagenesis without affecting therapeutic effectiveness

    Energy Technology Data Exchange (ETDEWEB)

    Kataoka, Yasushi [Univ. of Chicago, Chicago, IL (United States). Dept. of Radiation and Cellular Oncology; Perrin, J. [Argonne National Lab., IL (United States); Hunter, N.; Milas, L. [Univ. of Texas, Houston, TX (United States); Grdina, D. [Univ. of Chicago, Chicadgo, IL (United States). Dept. of Radiation and Cellular Oncology]|[Argonne National Lab., IL (United States)]|[Argonne National Lab., IL (United States)

    1995-12-31

    The radioprotector S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) was evaluated for its ability to protect against cytoxan-induced mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in mouse splenocytes under conditions that would not interfere with the therapeutic effectiveness of cytoxan in the treatment of fibrosarcoma lung tumors. Mutations at the hprt locus increase in frequency as a function of the dose of cytoxan used. With a spontaneous mutation frequency in C3H mice of 1.5 {times} 10{sup {minus}6}, mutation frequencies increased from 6.2 {times} 10{sup {minus}6} to 2.0 {times} 10{sup {minus}5} as the dose of cytoxan increased from 50 to 200 mg/kg. C3H male mice were injected in their tail veins with 3.5 {times} 10{sup 5} viable fibrosarcoma (FSa) cells. This protocol gave rise to an average of 68 tumor colonies per mouse. Four days following injection animals were treated with cytoxan at a dose of 100 mg/kg, which gave rise to significant tumor cell killing and a reduction in tumor colony number to less than an average of one per animal. WR-2721 at a concentration of 100 mg/kg did not affect on cytoxan`s therapeutic effectiveness. However, a 100 mg/kg dose of WR-2721 was effective in reducing the cytoxan induced hprt mutation frequency in mice from 160 to 35 per 10{sup 5} viable cells regardless of whether it was administered 30 min before or 2 h following cytoxan treatment.

  13. Phenotypic and biochemical profile changes in calendula (Calendula officinalis L.) plants treated with two chemical mutagenesis.

    Science.gov (United States)

    El-Nashar, Y I; Asrar, A A

    2016-05-06

    Chemical mutagenesis is an efficient tool used in mutation-breeding programs to improve the vital characters of the floricultural crops. This study aimed to estimate the effects of different concentrations of two chemical mutagens; sodium azide (SA) and diethyl sulfate (DES). The vegetative growth and flowering characteristics in two generations (M1 and M2) of calendula plants were investigated. Seeds were treated with five different concentrations of SA and DES (at the same rates) of 1000, 2000, 3000, 4000, and 5000 ppm, in addition to a control treatment of 0 ppm. Results showed that lower concentrations of SA mutagen had significant effects on seed germination percentage, plant height, leaf area, plant fresh weight, flowering date, inflorescence diameter, and gas-exchange measurements in plants of both generations. Calendula plants tended to flower earlier under low mutagen concentrations (1000 ppm), whereas higher concentrations delayed flowering significantly. Positive results on seed germination, plant height, number of branches, plant fresh weight, and leaf area were observed in the M2-generation at lower concentrations of SA (1000 ppm), as well as at 4000 ppm DES on number of leaves and inflorescences. The highest total soluble protein was detected at the concentrations of 1000 ppm SA and 2000 ppm DES. DES showed higher average of acid phosphatase activity than SA. Results indicated that lower concentrations of SA and DES mutagens had positive effects on seed germination percentage, plant height, leaf area, plant fresh weight, flowering date, inflorescence diameter, and gas-exchange measurements. Thus, lower mutagen concentrations could be recommended for better floral and physio-chemical performance.

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

    Directory of Open Access Journals (Sweden)

    Zhen Zhang

    2010-07-01

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

  15. Advances in knowledge of mutagenesis at the molecular level in plants

    International Nuclear Information System (INIS)

    Nilan, R.A.; Owais, W.M.; Rosichan, J.L.; Kleinhofs, A.

    1979-01-01

    The mechanism of action of the densely and sparsely ionizing types of radiation in plant cells has been investigated. The indirect actions of physical mutagens in plant cells through the modifying influences of oxygen, water content, and temperature are well known. In terms of the nature of the mutations induced by physical mutagens in plants, a vast array of mutational events can be induced by neutrons, X-ray, gamma-ray, etc. It was determined that Na azides have been highly efficient mutagen in barley, peas, soybean, maize, Chlamydomonas, rice, yeast, Chinese hamster cells and certain strains of S. typhimurium and E. coli. Na azides were used as respiration inhibitor to investigate how chromosomes break and mutations are induced and/or repaired in the cells of irradiated barley seeds. Azides alone in the presence of O 2 induced about 6% mutation of chlorophyll-deficient seedlings which could be increased to 20% when the seeds had been treated in azide solutions at the pH values below the pKa of azides. When the seeds were presoaked for 15 hours at 1 deg C and 12 - 16 hrs at 20 deg C in aerated solution prior to azide treatment, 75% mutation was obtained. This mutation frequency is compared to 17% and 40 - 45% induced in barley by X-ray and ethyl methanesulfonate, respectively. It was shown that both L-cysteine and L-cysteine inhibited azide mutagenesis, and the inhibition seemed to be specific to azides. The waxy locus of barley controlling the starch composition is used to measure forward and reverse mutations and mutant recombination frequency on the grain per million pollen basis, and provides high genetic resolution and a detailed map of the genes. (Yamashita, S.)

  16. Mechanisms of DNA repair and radio-induced mutagenesis in higher eukaryotes

    International Nuclear Information System (INIS)

    Averbeck, D.

    2000-01-01

    Cells of higher eukaryotes possess several very efficient systems for the repair of radiation-induced lesions in DNA. Different strategies have been adopted at the cellular level to remove or even tolerate various types of lesions in order to assure survival and limit the mutagenic consequences. In mammalian cells, the main DNA repair systems comprise direct reversion of damage, excision of damage and exchange mechanisms with intact DNA. Among these, the direct ligation of single strand breaks (SSB) by a DNA ligase and the multi-enzymatic repair systems of mismatch repair, base and nucleotide excision repair as well as the repair of double strand breaks (DSB) by homologous recombination or non homologous end-joining are the most important systems. Most of these processes are error-free except the non homologous end-joining pathway used for the repair of DSB. Moreover, certain lesions can be tolerated by more or less accurately acting polymerases capable of performing trans-lesion DNA syntheses. The DNA repair systems are intimately integrated in the network of cellular regulation. Some of their components are DNA damage inducible. Radiation-induced mutagenesis is largely due to unrepaired DNA damage but also involves error-prone repair processes like the repair of DSB by non-homologous end-joining. Generally, mammalian cells are well prepared to repair radiation-induced lesions. However, some questions remain to be asked about mechanistic details and efficiencies of the systems for removing certain types of radiation-damage and about their order and timing of action. The answers to these questions would be important for radioprotection as well as radiotherapy. (author)

  17. Principle and application of plant mutagenesis in crop improvement: a review

    Directory of Open Access Journals (Sweden)

    Yusuff Oladosu

    2016-01-01

    Full Text Available The first step in plant breeding is to identify suitable genotypes containing the desired genes among existing varieties, or to create one if it is not found in nature. In nature, variation occurs mainly as a result of mutations and without it, plant breeding would be impossible. In this context, the major aim in mutation-based breeding is to develop and improve well-adapted plant varieties by modifying one or two major traits to increase their productivity or quality. Both physical and chemical mutagenesis is used in inducing mutations in seeds and other planting materials. Then, selection for agronomic traits is done in the first generation, whereby most mutant lines may be discarded. The agronomic traits are confirmed in the second and third generations through evident phenotypic stability, while other evaluations are carried out in the subsequent generations. Finally, only the mutant lines with desirable traits are selected as a new variety or as a parent line for cross breeding. New varieties derived by induced mutatgenesis are used worldwide: rice in Vietnam, Thailand, China and the United States; durum wheat in Italy and Bulgaria; barley in Peru and European nations; soybean in Vietnam and China; wheat in China; as well as leguminous food crops in Pakistan and India. This paper integrates available data about the impact of mutation breeding-derived crop varieties around the world and highlights the potential of mutation breeding as a flexible and practicable approach applicable to any crop provided that appropriate objectives and selection methods are used.

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

    Directory of Open Access Journals (Sweden)

    Brian Healy

    Full Text Available 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.

  19. Gene transfer and genome-wide insertional mutagenesis by retroviral transduction in fish stem cells.

    Directory of Open Access Journals (Sweden)

    Qizhi Liu

    Full Text Available Retrovirus (RV is efficient for gene transfer and integration in dividing cells of diverse organisms. RV provides a powerful tool for insertional mutagenesis (IM to identify and functionally analyze genes essential for normal and pathological processes. Here we report RV-mediated gene transfer and genome-wide IM in fish stem cells from medaka and zebrafish. Three RVs were produced for fish cell transduction: rvLegfp and rvLcherry produce green fluorescent protein (GFP and mCherry fluorescent protein respectively under control of human cytomegalovirus immediate early promoter upon any chromosomal integration, whereas rvGTgfp contains a splicing acceptor and expresses GFP only upon gene trapping (GT via intronic in-frame integration and spliced to endogenous active genes. We show that rvLegfp and rvLcherry produce a transduction efficiency of 11~23% in medaka and zebrafish stem cell lines, which is as 30~67% efficient as the positive control in NIH/3T3. Upon co-infection with rvGTgfp and rvLcherry, GFP-positive cells were much fewer than Cherry-positive cells, consistent with rareness of productive gene trapping events versus random integration. Importantly, rvGTgfp infection in the medaka haploid embryonic stem (ES cell line HX1 generated GTgfp insertion on all 24 chromosomes of the haploid genome. Similar to the mammalian haploid cells, these insertion events were presented predominantly in intergenic regions and introns but rarely in exons. RV-transduced HX1 retained the ES cell properties such as stable growth, embryoid body formation and pluripotency gene expression. Therefore, RV is proficient for gene transfer and IM in fish stem cells. Our results open new avenue for genome-wide IM in medaka haploid ES cells in culture.

  20. Enhancement of Lutein Production in Chlorella sorokiniana (Chorophyta by Improvement of Culture Conditions and Random Mutagenesis

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    Maria Angeles Vargas

    2011-09-01

    Full Text Available Chlorella sorokiniana has been selected for lutein production, after a screening of thirteen species of microalgae, since it showed both a high content in this carotenoid and a high growth rate. The effects of several nutritional and environmental factors on cell growth and lutein accumulation have been studied. Maximal specific growth rate and lutein content were attained at 690 µmol photons m−2 s−1, 28 °C, 2 mM NaCl, 40 mM nitrate and under mixotrophic conditions. In general, optimal conditions for the growth of this strain also lead to maximal lutein productivity. High lutein yielding mutants of C. sorokiniana have been obtained by random mutagenesis, using N-methyl-N′-nitro-nitrosoguanidine (MNNG as a mutagen and selecting mutants by their resistance to the inhibitors of the carotenogenic pathway nicotine and norflurazon. Among the mutants resistant to the herbicides, those exhibiting both high content in lutein and high growth rate were chosen. Several mutants exhibited higher contents in this carotenoid than the wild type, showing, in addition, either a similar or higher growth rate than the latter strain. The mutant MR-16 exhibited a 2.0-fold higher volumetric lutein content than that of the wild type, attaining values of 42.0 mg L−1 and mutants DMR-5 and DMR-8 attained a lutein cellular content of 7.0 mg g−1 dry weight. The high lutein yield exhibited by C. sorokiniana makes this microalga an excellent candidate for the production of this commercially interesting pigment.