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Sample records for site-directed mutagenesis short

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

    Indian Academy of Sciences (India)

    In this article, the traditional multi-site-directed mutagenesis method based on overlap extension PCR was improved specifically for complicated templates, such as genomic sequence or complementary DNA. This method was effectively applied for multi-site-directed mutagenesis directly from mouse genomic DNA, as well ...

  2. 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.

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

    Science.gov (United States)

    Atanassov, Ivan I; Atanassov, Ilian I; Etchells, J Peter; Turner, Simon R

    2009-01-01

    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. PMID:19863796

  4. 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 ...

  5. Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h

    OpenAIRE

    Chiu, Joyce; March, Paul E.; Lee, Ryan; Tillett, Daniel

    2004-01-01

    Site-directed, Ligase-Independent Mutagenesis (SLIM) is a novel PCR-mediated mutagenesis approach that can accommodate all three sequence modification types (insertion, deletion and substitution). The method utilizes an inverse PCR amplification of the template by two tailed long primers and two short primers in a single reaction with all steps carried out in one tube. The tailed primers are designed to contain the desired mutation on complementary overhangs at the terminus of PCR products. U...

  6. 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...

  7. Ultrafast solvation dynamics at internal site of staphylococcal nuclease investigated by site-directed mutagenesis

    CERN Document Server

    Guang-yu, Gao; Wei, Wang; Shu-feng, Wang; Zhong, Dongping; Qi-huang, Gong

    2014-01-01

    Solvation is essential for protein activities. To study internal solvation of protein, site-directed mutagenesis is applied. Intrinsic fluorescent probe, tryptophan, is inserted into desired position inside protein molecule for ultrafast spectroscopic study. Here we review this unique method for protein dynamics researches. We introduce the frontiers of protein solvation, site-directed mutagenesis, protein stability and characteristics, and the spectroscopic methods. Then we present time-resolved spectroscopic dynamics of solvation dynamics inside caves of active sites. The studies are carried out on a globular protein, staphylococcal nuclease. The solvation at internal sites of the caves indicate clear characteristics of local environment. These solvation behaviors correlated to the enzyme activity directly.

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

    National Research Council Canada - National Science Library

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

    2016-01-01

    .... Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins...

  9. Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h.

    Science.gov (United States)

    Chiu, Joyce; March, Paul E; Lee, Ryan; Tillett, Daniel

    2004-12-07

    Site-directed, Ligase-Independent Mutagenesis (SLIM) is a novel PCR-mediated mutagenesis approach that can accommodate all three sequence modification types (insertion, deletion and substitution). The method utilizes an inverse PCR amplification of the template by two tailed long primers and two short primers in a single reaction with all steps carried out in one tube. The tailed primers are designed to contain the desired mutation on complementary overhangs at the terminus of PCR products. Upon post-amplification denaturation and re-annealing, heteroduplex formation between the mixed PCR products creates the desired clonable mutated plasmid. The technique is highly robust and suitable for applications in high-throughput gene engineering and library constructions. In this study, SLIM was employed to create sequence insertions, deletion and substitution within bacteriophage T7 gene 5. The overall efficiency for obtaining the desired product was >95%.

  10. 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.

  11. Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    Science.gov (United States)

    Weng, Meizhi; Deng, Xiongwei; Bao, Wei; Zhu, Li; Wu, Jieyuan; Cai, Yongjun; Jia, Yan; Zheng, Zhongliang; Zou, Guolin

    2015-09-25

    Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Site-directed mutagenesis of substrate binding sites of azoreductase from Rhodobacter sphaeroides.

    Science.gov (United States)

    Liu, Guangfei; Zhou, Jiti; Wang, Jing; Yan, Bin; Li, Jingmei; Lu, Hong; Qu, Yuanyuan; Jin, Ruofei

    2008-05-01

    Comparison of three-dimensional structures of flavin-dependent azoreductases revealed two conserved loops around the flavin mononucleotide (FMN) cofactor. Tyr74, His75 and Lys109 in the two loops of azoreductase AZR from Rhodobacter sphaeroides were replaced with Trp, Asn and Ala/His by site-directed mutagenesis, respectively. The optimal pH values of K109H and H75N were pH 6, and those of K109A and Y74W were pH 9. The optimal temperature (30 degrees C) was not affected by mutation. Positively charged residues at position 109 is critical for the binding of methyl red. K109 might only be involved in the binding of the 2'-phosphate group of NADPH and have no effect on the binding of NADH. Y74W and H75N mutations decreased the binding of methyl red/nitrofurazone and had no affect on the binding of NADPH.

  13. Crystal structure and site-directed mutagenesis of a nitroalkane oxidase from Streptomyces ansochromogenes.

    Science.gov (United States)

    Li, Yanhua; Gao, Zengqiang; Hou, Haifeng; Li, Lei; Zhang, Jihui; Yang, Haihua; Dong, Yuhui; Tan, Huarong

    2011-02-18

    Nitroalkane oxidase (NAO) catalyzes neutral nitroalkanes to their corresponding aldehydes or ketones, hydrogen peroxide and nitrite. The crystal structure of NAO from Streptomyces ansochromogenes was determined; it consists of two domains, a TIM barrel domain bound to FMN and C-terminal domain with a novel folding pattern. Site-directed mutagenesis of His179, which is spatially adjacent to FMN, resulted in the loss of enzyme activity, demonstrating that this amino acid residue is important for catalysis. The crystal structure of mutant H179D-nitroethane was also analyzed. Interestingly, Sa-NAO shows the typical function as nitroalkane oxidase but its structure is similar to that of 2-nitropropane dioxygenase. Overall, these results suggest that Sa-NAO is a novel nitroalkane oxidase with TIM barrel structure. Copyright © 2010 Elsevier Inc. All rights reserved.

  14. Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System.

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    Audrey Olshefsky

    Full Text Available Two-component signaling (2CS systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli's natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or "coliroids," rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by β-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein's local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system's performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling.

  15. Increased activity of β-glucuronidase variants produced by site-directed mutagenesis.

    Science.gov (United States)

    Zhang, Xiaolei; Sitasuwan, Pongkwan; Horvath, Gary; Yang, Jia; Nie, Yuzhe; Marinova, Margarita; Lee, L Andrew; Wang, Qian

    2018-02-01

    β-glucuronidase (BGus) is an essential glycosyl hydrolase which has been widely used in biological and biomedical applications. In this paper, we report the construction and screening of nineteen Escherichia coli BGus (EBGus) mutants using site-directed mutagenesis. The mutants G559N, G559S and G559T showed a 3-5 fold increase in enzyme activity in comparison to wild type EBGus. In particular, G559S, with the highest activity, showed 2-6 fold enhanced activity compared to abalone and snail BGus extracts. Moreover, the glycine to serine mutagenesis for the same site in Staphylococcus sp. RLH1 BGus (StBGus) exhibited significantly enhanced activity, which indicated the importance of the G559→S mutation on BGus function. Based on this structural analysis, we postulate that the mutation at G559 plays an important role in the stabilization of the enzyme conformation, and thereby facilitates the effective binding of substrate. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. 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 T1/2, 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.

  17. 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.

  18. Improving the Thermostability of Raw-Starch-Digesting Amylase from a Cytophaga sp. by Site-Directed Mutagenesis

    OpenAIRE

    Shiau, Rong-Jen; Hung, Hui-Chen; Jeang, Chii-Ling

    2003-01-01

    A heat-stable raw-starch-digesting amylase (RSDA) was generated through PCR-based site-directed mutagenesis. At 65°C, the half-life of this mutant RSDA, which, compared with the wild-type RSDA, lacks amino acids R178 and G179, was increased 20-fold. While the wild type was inactivated completely at pH 3.0, the mutant RSDA still retained 41% of its enzymatic activity. The enhancement of RSDA thermostability was demonstrated to be via a Ca2+-independent mechanism.

  19. Improving the Thermostability of Raw-Starch-Digesting Amylase from a Cytophaga sp. by Site-Directed Mutagenesis

    Science.gov (United States)

    Shiau, Rong-Jen; Hung, Hui-Chen; Jeang, Chii-Ling

    2003-01-01

    A heat-stable raw-starch-digesting amylase (RSDA) was generated through PCR-based site-directed mutagenesis. At 65°C, the half-life of this mutant RSDA, which, compared with the wild-type RSDA, lacks amino acids R178 and G179, was increased 20-fold. While the wild type was inactivated completely at pH 3.0, the mutant RSDA still retained 41% of its enzymatic activity. The enhancement of RSDA thermostability was demonstrated to be via a Ca2+-independent mechanism. PMID:12676725

  20. The roles of cytochrome b559 in assembly and photoprotection of Photosystem II revealed by site-directed mutagenesis studies

    Directory of Open Access Journals (Sweden)

    Hsiu-An eChu

    2016-01-01

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

  1. Enhancement of oxidative stability of the subtilisin nattokinase by site-directed mutagenesis expressed in Escherichia coli.

    Science.gov (United States)

    Weng, MeiZhi; Zheng, ZhongLiang; Bao, Wei; Cai, YongJun; Yin, Yan; Zou, GuoLin; Zou, GouLin

    2009-11-01

    Nattokinase (subtilisin NAT, NK) is a bacterial serine protease with strong fibrinolytic activity and it is a potent cardiovascular drug. In medical and commercial applications, however, it is susceptible to chemical oxidation, and subsequent inactivation or denaturation. Here we show that the oxidative stability of NK was substantially increased by optimizing the amino acid residues Thr(220) and Met(222), which were in the vicinity of the catalytic residue Ser(221) of the enzyme. Two nonoxidative amino acids (Ser and Ala) were introduced at these sites using site-directed mutagenesis. Active enzymes were successfully expressed in Escherichia coli with periplasmic secretion and enzymes were purified to homogeneity. The purified enzymes were analyzed with respect to oxidative stability, kinetic parameters, fibrinolytic activity and thermal stability. M222A mutant was found to have a greatly increased oxidative stability compared with wild-type enzyme and it was resistant to inactivation by more than 1 M H(2)O(2), whereas the wild-type enzyme was inactivated by 0.1 M H(2)O(2) (t(1/2) approximately 11.6 min). The other mutant (T220S) also showed an obvious increase in antioxidative ability. Molecular dynamic simulations on wild-type and T220S mutant proteins suggested that a hydrogen bond was formed between Ser(220) and Asn(155), and the spatial structure of Met(222) was changed compared with the wild-type. The present study demonstrates the feasibility of improving oxidative stability of NK by site-directed mutagenesis and shows successful protein engineering cases to improve stability of NK as a potent therapeutic agent.

  2. Functional expression enhancement of Bacillus pumilus CotA-laccase mutant WLF through site-directed mutagenesis.

    Science.gov (United States)

    Luo, Quan; Chen, Yu; Xia, Jing; Wang, Kai-Qiang; Cai, Yu-Jie; Liao, Xiang-Ru; Guan, Zheng-Bing

    2018-02-01

    Bacterial laccases are potential enzymes for biotechnological applications, such as detoxification of industrial effluents, decolorization of textile, and dimerization of phenolic acids, due to their remarkable advantages, including broad substrate spectrum, high thermostability, wide pH scope, and tolerance to alkaline environments. L386W/G417L/G57F (abbreviated as WLF), a good mutant of CotA-laccase from Bacillus pumilus W3, has been constructed and reported by our laboratory with highly improved catalytic efficiency. However, the low-functional expression level of mutant WLF in Escherichia coli was a shortcoming. Three mutants, namely, K317N/WLF, D501G/WLF, and K317N/D501G/WLF, were constructed through site-directed mutagenesis to improve the functional expression of WLF in this study. The soluble and active expression of D501G/WLF and K317N/D501G/WLF in E. coli enhanced 4.48-fold and 3.63-fold level, respectively. The K317N/WLF failed to increase the soluble expression level, but slightly improved the stability of CotA-laccase. Results showed that not only the position 501 is significant for functional expression of B. pumilus W3 CotA, but also these mutants still remained its high thermostability, resistance of alkaline with salt, and conspicuous decolorizing efficiency. This work is the first to improve the soluble expression of B. pumilus CotA-laccase in E. coli by site-directed mutagenesis. The D501G/WLF and K317N/D501G/WLF will be suitable candidates for biotechnological applications. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2015-01-01

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

  4. Identification of residues involved in nucleotidyltransferase activity of JHP933 from helicobacter pyloriby site-directed mutagenesis

    Directory of Open Access Journals (Sweden)

    Ye Xianren

    2016-01-01

    Full Text Available Helicobacter pylori is a well-known bacterial pathogen involved in the development of peptic ulcer, gastric adenocarcinoma and other forms of gastric cancer. Evidence has suggested that certain strain-specific genes in the plasticity region may play key roles in the pathogenesis of H. pylori-associated gastroduodenal diseases. Therefore there is considerable interest in the strain-specific genes located in the plasticity regions of H. pylori. JHP933 is encoded by the gene in the plasticity region of H. pylori strain J99. Recently, the crystal structure of JHP933 has confirmed it as a nucleotidyltransferase (NTase superfamily protein and a putative active site has been proposed. However, no evidence from direct functional assay has been presented to confirm the active site and little is known about the functional mechanism of JHP933. Here, through superimposition with Cid1/NTP complex structures, we modelled the complex structures of JHP933 with different NTPs. Based on the models and using rational site-directed mutagenesis combined with enzymatic activity assays, we confirm the active site and identify several residues important for the nucleotidyl transferring function of JHP933. Furthermore, mutations of these active site residues result in the abolishment of the nucleotidyltransferase activity of JHP933. This work provides preliminary insight into the molecular mechanism underlying the pathophysiological role in H. pylori infection of JHP933 as a novel NTase superfamily protein.

  5. 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.

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

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    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.

  7. 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.

  8. Partial identification by site-directed mutagenesis of a cell growth inhibitory site on the human galectin-1 molecule

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    Zhang Jialiang

    2002-01-01

    Full Text Available Abstract Background Previous work, by us and others, has shown that mammalian galectins-1 have a growth-inhibitory activity for mammalian cells which is apparently independent of their β-galactoside binding site. Results We have made recombinant human galectin-1 as a bacterial fusion protein with an N-terminal hexahistidine tag. This protein displays both haemagglutination and growth-inhibitory activities, even in the presence of the hexahistidine tag. Site-directed mutagenesis of this protein has confirmed the independent nature of the protein sites responsible for the two biological activities. Mutant proteins were created, which displayed each activity in the absence of the other. Conclusions Human galectin-1 possesses a growth-inhibitory site, which is not part of the β-galactoside binding site. A surface loop, comprising amino acid residues 25–30, and joining two internal β-strands, forms part of the growth-inhibitory site. This region is relatively close to the N-terminus of the protein, and N-terminal substitutions or extensions also affect growth-inhibitory activity. Further experiments will be necessary to fully define this site.

  9. 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.

  10. Site-directed mutagenesis and footprinting analysis of the interaction of the sunflower KNOX protein HAKN1 with DNA.

    Science.gov (United States)

    Tioni, Mariana F; Viola, Ivana L; Chan, Raquel L; Gonzalez, Daniel H

    2005-01-01

    The interaction of the homeodomain of the sunflower KNOX protein HAKN1 with DNA was studied by site-directed mutagenesis, hydroxyl radical footprinting and missing nucleoside experiments. Binding of HAKN1 to different oligonucleotides indicated that HAKN1 prefers the sequence TGACA (TGTCA), with changes within the GAC core more profoundly affecting the interaction. Footprinting and missing nucleoside experiments using hydroxyl radical cleavage of DNA showed that HAKN1 interacts with a 6-bp region of the strand carrying the GAC core, covering the core and nucleotides towards the 3' end. On the other strand, protection was observed along an 8-bp region, comprising two additional nucleotides complementary to those preceding the core. Changes in the residue present at position 50 produced proteins with different specificities. An I50S mutant showed a preference for TGACT, while the presence of lysine shifted the preference to TGACC, suggesting that residue 50 interacts with nucleotide(s) 3' to GAC. Mutation of Lys54-->Val produced a protein with reduced affinity and relaxed specificity, able to recognize the sequence TGAAA, while the conservative change of Arg55-->Lys completely abolished binding to DNA. Based on these results, we propose a model for the interaction of HAKN1 with DNA in which helix III of the homeodomain accommodates along the major groove with Arg55, Asn51, Lys54 and Ile50, establishing specific contacts with bases of the GACA sequence or their complements. This model can be extended to other KNOX proteins given the conservation of these amino acids in all members of the family.

  11. Proton transfers in a channelrhodopsin-1 studied by Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis.

    Science.gov (United States)

    Ogren, John I; Yi, Adrian; Mamaev, Sergey; Li, Hai; Spudich, John L; Rothschild, Kenneth J

    2015-05-15

    Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. 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.

  13. Site-directed mutagenesis of human immunodeficiency virus type 1 reverse transcriptase at amino acid position 138.

    Science.gov (United States)

    Pelemans, H; Aertsen, A; Van Laethem, K; Vandamme, A M; De Clercq, E; Pérez-Pérez, M J; San-Félix, A; Velázquez, S; Camarasa, M J; Balzarini, J

    2001-02-01

    TSAO derivatives represent a class of nonnucleoside reverse transcriptase inhibitors (NNRTIs) that consistently select for the Glu138Lys resistance mutation in HIV-1 reverse transcriptase (RT). Seven RT mutants (i.e., Ala, Asp, Gln, Gly, Lys, Phe, and Tyr) were constructed by site-directed mutagenesis. The mutant Glu138Asp, Glu138Lys, Glu138Gln, Glu138Ala, and Glu138Gly RTs retained marked catalytic activity. In contrast, the Glu138Phe and Glu138Tyr RT mutants showed poor RNA-dependent DNA polymerase activity (30 and 4% of wild-type, respectively). TSAO derivatives lost their inhibitory activity against all mutant enzymes, except against the closely related Glu138Asp RT mutant that remained as sensitive to TSAOs as did wild-type RT. Other NNRTIs, including delavirdine, emivirine, and UC-781, and the NRTI ddGTP retained pronounced inhibitory activity against all mutant enzymes. When the amino acid mutations at position 138 of RT were introduced in recombinant virus clones, the sensitivity/resistance spectrum obtained toward the TSAOs and other NNRTIs was similar to those observed for the isolated recombinant mutant enzymes. The Glu138Lys RT mutant virus had the most marked resistance to TSAOs, followed by the Glu138Gln, Glu138Phe, Glu138Gly, Glu138Tyr, and Glu138Ala virus mutants. The Glu138Asp RT mutant virus kept full sensitivity to the TSAO derivatives. Mixtures of Glu138Lys RT mutant virus with the other virus clones mutated at the 138 position resulted in all cases, except for the Glu138Asp and Glu138Gly RT mutant viruses, in an outgrowth of the Glu138Lys RT mutant virus. Since the Glu138Lys RT proved most resistant to TSAO derivatives, was among the most catalytically efficient enzymes, and resulted in highly replication-competent virus, our data explain why the Glu138Lys RT mutant virus strains but not virus strains containing other amino acids at position 138 invariably emerge in cell cultures under TSAO drug pressure. Copyright 2001 Academic Press.

  14. Effects of site-directed mutagenesis in the N-terminal domain of thermolysin on its stabilization.

    Science.gov (United States)

    Kawasaki, Yuichi; Yasukawa, Kiyoshi; Inouye, Kuniyo

    2013-01-01

    The thermolysin variant G8C/N60C/S65P in which the triple mutation in the N-terminal domain, Gly8→Cys/Asn60→Cys/Ser65→Pro, is undertaken increases stability [Yasukawa, K. and Inouye, K. (2007) Improving the activity and stability of thermolysin by site-directed mutagenesis. Biochim. Biophys. Acta 1774, 1281-1288] and its mechanism is examined in this study. The apparent denaturing temperatures based on ellipticity at 222 nm of the wild-type thermolysin (WT), G8C/N60C, S65P and G8C/N60C/S65P were 85, >95, 88 and >95°C, respectively. The first-order rate constants, k(obs), of the thermal inactivation of WT and variants at 10 mM CaCl₂ increased with increasing thermal treatment temperatures (70-95°C), and those at 80°C decreased with increasing CaCl₂ concentrations (1-100 mM). The k(obs) values were in the order of WT > S65P > G8C/N60C≒G8C/N60C/S65P at all temperatures and CaCl₂ concentrations. These results indicate that the mutational combination, Gly8→Cys/Asn60→Cys and Ser65→Pro, increases stability only as high as Gly8→Cys/Asn60→Cys does. Assuming that irreversible inactivation of thermolysin occurs only in the absence of calcium ions, the dissociation constants, K(d), to the calcium ions of WT, G8C/N60C, S65P and G8C/N60C/S65P were 47, 8.9, 17 and 7.2 mM, respectively, suggesting that Gly8→Cys/Asn60→Cys and Ser65→Pro stabilize thermolysin by improving its affinity to calcium ions, most probably the one at the Ca²⁺-binding site III in the N-terminal domain.

  15. 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…

  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-NPm-8, PP-NPep-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-NPep-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-NPep-6A, PP-NPm-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-NPep-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, 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

  18. Attempting to remove the substrate inhibition of L-lactate dehydrogenase from Bacillus stearothermophilus by site-directed mutagenesis.

    Science.gov (United States)

    Binay, Bariş; Karagüler, Nevin Gül

    2007-01-01

    L-lactate dehydrogenase (LDH) catalyzes the interconversion of an oxoacid (pyruvate) and hydroxy-acid (lactate) using the NADH/NAD+ pair as a redox cofactor. The enzyme has a commercial significance, as it can be used to produce chiral building blocks for the synthesis of key pharmaceuticals and agrochemicals. However, the substrate inhibition which is due to an abortive NAD+-pyruvate complex reducing the steady state concentration of functional LDH limits its use in industry. This substrate inhibition can be overcome by weaking the binding of NAD+. The conserved aspartic acid residue at position 38 was replaced by the longer basic arginine side chain (D38R) using PCR based overlap extension mutagenesis technique in the hope of weakening NAD+-binding. The mutant gene was overexpressed in the Escherichia coli high-expression vector pKK223-3 in JM105 cells; then, the mutant protein was produced. Comparing the effect of substrate inhibition in the arginine-38 mutant with wild-type, substrate inhibition is decreased threefold.

  19. Probing the specificity of the subclass B3 FEZ-1 metallo-beta-lactamase by site-directed mutagenesis.

    Science.gov (United States)

    Mercuri, Paola Sandra; García-Sáez, Isabel; De Vriendt, Kris; Thamm, Iris; Devreese, Bart; Van Beeumen, Jozef; Dideberg, Otto; Rossolini, Gian Maria; Frère, Jean-Marie; Galleni, Moreno

    2004-08-06

    The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography. In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics. The activity of H121A is dependent on zinc ion concentration. The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme. Tyr156 is not essential for binding and hydrolysis of the substrate. Substitution of residues Ser221 and Asn225 modifies the substrate profile by selectively decreasing the activity against carbapenems. The Y228A mutant is inhibited by the product formed upon hydrolysis of cephalosporins. A covalent bond between the side chain of Cys200 and the hydrolyzed cephalosporins leads to the formation of an inactive and stable complex.

  20. Site-directed mutagenesis around the CuA site of a polyphenol oxidase from Coreopsis grandiflora (cgAUS1).

    Science.gov (United States)

    Kaintz, Cornelia; Mayer, Rupert L; Jirsa, Franz; Halbwirth, Heidi; Rompel, Annette

    2015-03-24

    Aurone synthase from Coreopsis grandiflora (cgAUS1), catalyzing conversion of butein to sulfuretin in a type-3 copper center, is a rare example of a polyphenol oxidase involved in anabolism. Site-directed mutagenesis around the CuA site of AUS1 was performed, and recombinant enzymes were analyzed by mass spectrometry. Replacement of the coordinating CuA histidines with alanine resulted in the presence of a single copper and loss of diphenolase activity. The thioether bridge-building cysteine and a phenylalanine over the CuA site, exchanged to alanine, have no influence on copper content but appear to play an important role in substrate binding. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  1. 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

    using site-directed mutagenesis. Recombinants were also generated using PCR-amplified ftsI from clinical strains encoding multiple amino acid substitutions. MICs of ampicillin, cefuroxime, cefotaxime and ceftriaxone were determined using Etest ® . Results: Transformation of a susceptible strain with fts......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...

  2. 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 (Chi960235-459) containing the substrate-binding domain using the homologous 1ITX protein of Bacillus circulans as the template. We performed molecular docking analysis of Chi960235-459 using di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli. Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several plant-pathogenic fungi were observed. PMID:25678849

  3. 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.

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

    Science.gov (United States)

    Reinstädler, Anja; Müller, Judith; Czembor, Jerzy H; Piffanelli, Pietro; Panstruga, Ralph

    2010-02-19

    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. 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. 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.

  5. 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.

  6. Site directed mutagenesis experiments suggest that Glu 111, Glu 144 and Arg 145 are essential for endonucleolytic activity of EcoRI.

    Science.gov (United States)

    Wolfes, H; Alves, J; Fliess, A; Geiger, R; Pingoud, A

    1986-11-25

    We have constructed a plasmid (pRIF 309+) carrying the EcoRI restriction endonuclease gene and the f1 origin of replication. Upon transformation of this plasmid into E. coli and infection with bacteriophage f1 single stranded plasmids are produced which can be used for sequencing and site directed mutagenesis. Using this single stranded DNA and synthetic oligodeoxynucleotides we have introduced point mutations at defined positions of the EcoRI gene. Since in pRIF309+ the EcoRI gene is under the control of the pL-promoter, high level expression of the mutated EcoRI gene could be obtained upon induction. Mutant EcoRI enzymes were purified to homogeneity and characterized in structural and functional terms. Our results demonstrate that the Glu 111----Gln, Glu 144----Gln and Arg 145----Lys -mutants adopt a very similar conformation as the wild type enzyme, but have by two orders of magnitude smaller specific activities than the wild type enzyme, mainly due to a reduction of the Vmax-value.

  7. 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.

  8. Toward an understanding of agonist binding to human Orexin-1 and Orexin-2 receptors with G-protein-coupled receptor modeling and site-directed mutagenesis.

    Science.gov (United States)

    Heifetz, Alexander; Barker, Oliver; Morris, G Benjamin; Law, Richard J; Slack, Mark; Biggin, Philip C

    2013-11-19

    The class A G-protein-coupled receptors (GPCRs) Orexin-1 (OX1) and Orexin-2 (OX2) are located predominantly in the brain and are linked to a range of different physiological functions, including the control of feeding, energy metabolism, modulation of neuro-endocrine function, and regulation of the sleep-wake cycle. The natural agonists for OX1 and OX2 are two neuropeptides, Orexin-A and Orexin-B, which have activity at both receptors. Site-directed mutagenesis (SDM) has been reported on both the receptors and the peptides and has provided important insight into key features responsible for agonist activity. However, the structural interpretation of how these data are linked together is still lacking. In this work, we produced and used SDM data, homology modeling followed by MD simulation, and ensemble-flexible docking to generate binding poses of the Orexin peptides in the OX receptors to rationalize the SDM data. We also developed a protein pairwise similarity comparing method (ProS) and a GPCR-likeness assessment score (GLAS) to explore the structural data generated within a molecular dynamics simulation and to help distinguish between different GPCR substates. The results demonstrate how these newly developed methods of structural assessment for GPCRs can be used to provide a working model of neuropeptide-Orexin receptor interaction.

  9. Essentiality of Lys-329 of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum as demonstrated by site-directed mutagenesis.

    Science.gov (United States)

    Soper, T S; Mural, R J; Larimer, F W; Lee, E H; Machanoff, R; Hartman, F C

    1988-04-01

    The unusual chemical properties of active-site Lys-329 of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have suggested that this residue is required for catalysis. To test this postulate Lys-329 was replaced with glycine, serine, alanine, cysteine, arginine, glutamic acid or glutamine by site-directed mutagenesis. These single amino acid substitutions do not appear to induce major conformational changes because (i) intersubunit interactions are unperturbed in that the purified mutant proteins are stable dimers like the wild-type enzyme and (ii) intrasubunit folding is normal in that the mutant proteins bind the competitive inhibitor 6-phosphogluconate with an affinity similar to that of wild-type enzyme. In contrast, all of the mutant proteins are severely deficient in carboxylase activity (less than 0.01% of wild-type) and are unable to form the exchange-inert complex, characteristic of the wild-type enzyme, with the transition-state analogue carboxyarabinitol bisphosphate. These results underscore the stringency of the requirement for a lysyl side-chain at position 329 and imply that Lys-329 is involved in catalysis, perhaps stabilizing a transition state in the overall reaction pathway.

  10. Examination of the intersubunit interaction between glutamate-48 and lysine-168 of ribulose-bisphosphate carboxylase/oxygenase by site-directed mutagenesis.

    Science.gov (United States)

    Mural, R J; Soper, T S; Larimer, F W; Hartman, F C

    1990-04-15

    The active site of ribulose-bisphosphate carboxylase/oxygenase is constituted from domains of adjacent subunits and includes an intersubunit electrostatic interaction between Lys 168 and Glu48, which has been recently identified by x-ray crystallography (Andersson, I., Knight, S., Schneider, G., Lindqvist, Y., Lundqvist, T., Brändén, C.-I., and Lorimer, G.H. (1989) Nature 337, 229-234; Lundqvist, T., and Schneider, G. (1989) J. Biol. Chem. 264, 7078-7083). To examine the structural and functional requirements for this interaction, we have used site-directed mutagenesis to replace Lys168 of the homodimeric enzyme from Rhodospirillum rubrum with arginine, glutamine, or glutamic acid. All three substitutions result in mutant enzymes with less than or equal to 0.1% of wild-type activity. The nonconservative substitution of Lys168 with a glutamyl residue precludes the formation of a stable dimer, explaining the consequential abolition of enzymic activity. Both the Arg168 and Gln168 mutant proteins are isolated as stable dimers, even though the latter obviously lacks an electrostatic interaction present in the wild-type enzyme. Despite the absence of overall carboxylase activity, these two mutant proteins serve as catalysts for the enolization of ribulose bisphosphate, as measured by exchange of the C3 proton with solvent. These observations, as well as ligand-binding properties of the mutant proteins, are consistent with Lys168 facilitating a catalytic step subsequent to enolization.

  11. Mechanism-based site-directed mutagenesis to shift the optimum pH of the phenylalanine ammonia-lyase from Rhodotorula glutinis JN-1

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    Longbao Zhu

    2014-09-01

    Full Text Available Phenylalanine ammonia-lyase (RgPAL from Rhodotorula glutinis JN-1 stereoselectively catalyzes the conversion of the l-phenylalanine into trans-cinnamic acid and ammonia, and was used in chiral resolution of dl-phenylalanine to produce the d-phenylalanine under acidic condition. However, the optimum pH of RgPAL is 9 and the RgPAL exhibits low catalytic efficiency at acidic side. Therefore, a mutant RgPAL with a lower optimum pH is expected. Based on catalytic mechanism and structure analysis, we constructed a mutant RgPAL-Q137E by site-directed mutagenesis, and found that this mutant had an extended optimum pH 7–9 with activity of 1.8-fold higher than that of the wild type at pH 7. As revealed by Friedel–Crafts-type mechanism of RgPAL, the improvement of the RgPAL-Q137E might be due to the negative charge of Glu137 which could stabilize the intermediate transition states through electrostatic interaction. The RgPAL-Q137E mutant was used to resolve the racemic dl-phenylalanine, and the conversion rate and the eeD value of d-phenylalanine using RgPAL-Q137E at pH 7 were increased by 29% and 48%, and achieved 93% and 86%, respectively. This work provides an effective strategy to shift the optimum pH which is favorable to further applications of RgPAL.

  12. Mechanism-based site-directed mutagenesis to shift the optimum pH of the phenylalanine ammonia-lyase from Rhodotorula glutinis JN-1.

    Science.gov (United States)

    Zhu, Longbao; Zhou, Li; Cui, Wenjing; Liu, Zhongmei; Zhou, Zhemin

    2014-09-01

    Phenylalanine ammonia-lyase (RgPAL) from Rhodotorula glutinis JN-1 stereoselectively catalyzes the conversion of the l-phenylalanine into trans-cinnamic acid and ammonia, and was used in chiral resolution of dl-phenylalanine to produce the d-phenylalanine under acidic condition. However, the optimum pH of RgPAL is 9 and the RgPAL exhibits low catalytic efficiency at acidic side. Therefore, a mutant RgPAL with a lower optimum pH is expected. Based on catalytic mechanism and structure analysis, we constructed a mutant RgPAL-Q137E by site-directed mutagenesis, and found that this mutant had an extended optimum pH 7-9 with activity of 1.8-fold higher than that of the wild type at pH 7. As revealed by Friedel-Crafts-type mechanism of RgPAL, the improvement of the RgPAL-Q137E might be due to the negative charge of Glu137 which could stabilize the intermediate transition states through electrostatic interaction. The RgPAL-Q137E mutant was used to resolve the racemic dl-phenylalanine, and the conversion rate and the eeD value of d-phenylalanine using RgPAL-Q137E at pH 7 were increased by 29% and 48%, and achieved 93% and 86%, respectively. This work provides an effective strategy to shift the optimum pH which is favorable to further applications of RgPAL.

  13. Mapping the Anopheles gambiae odorant binding protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays.

    Science.gov (United States)

    Rusconi, B; Maranhao, A C; Fuhrer, J P; Krotee, P; Choi, S H; Grun, F; Thireou, T; Dimitratos, S D; Woods, D F; Marinotti, O; Walter, M F; Eliopoulos, E

    2012-08-01

    The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Mapping the Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays

    Science.gov (United States)

    Rusconi, B; Maranhao, AC; Fuhrer, JP; Krotee, P; Choi, SH; Grun, F; Thireou, T; Dimitratos, SD; Woods, DF; Marinotti, O; Walter, MF; Eliopoulos, E

    2012-01-01

    The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to 22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics. PMID:22564768

  15. Human peroxisomal multifunctional enzyme type 2. Site-directed mutagenesis studies show the importance of two protic residues for 2-enoyl-CoA hydratase 2 activity.

    Science.gov (United States)

    Qin, Y M; Haapalainen, A M; Kilpeläinen, S H; Marttila, M S; Koski, M K; Glumoff, T; Novikov, D K; Hiltunen, J K

    2000-02-18

    Beta-oxidation of acyl-CoAs in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. Amino acid sequence alignment of the 2-enoyl-CoA hydratase 2 domain in human MFE-2 with other MFE-2s reveals conserved protic residues: Tyr-347, Glu-366, Asp-370, His-406, Glu-408, Tyr-410, Asp-490, Tyr-505, Asp-510, His-515, Asp-517, and His-532. To investigate their potential roles in catalysis, each residue was replaced by alanine in site-directed mutagenesis, and the resulting constructs were tested for complementation in a yeast. After additional screening, the wild type and noncomplementing E366A and D510A variants were expressed and characterized. The purified proteins have similar secondary structural elements, with the same subunit composition. The E366A variant had a k(cat)/K(m) value 100 times lower than that of the wild type MFE-2 at pH 5, whereas the D510A variant was inactive. Asp-510 was imbedded in a novel hydratase 2 motif found in the hydratase 2 proteins. The data show that the hydratase 2 reaction catalyzed by MFE-2 requires two protic residues, Glu-366 and Asp-510, suggesting that their catalytic role may be equivalent to that of the two catalytic residues of hydratase 1.

  16. Site-directed mutagenesis of the Arabidopsis heterotrimeric G protein β subunit suggests divergent mechanisms of effector activation between plant and animal G proteins.

    Science.gov (United States)

    Chakravorty, David; Trusov, Yuri; Botella, José Ramón

    2012-03-01

    Heterotrimeric G proteins are integral components of signal transduction in humans and other mammals and have been therefore extensively studied. However, while they are known to mediate many processes, much less is currently known about the effector pathways and molecular mechanisms used by these proteins to regulate effectors in plants. We designed a complementation strategy to study G protein signaling in Arabidopsis thaliana, particularly the mechanism of action of AGB1, the sole identified β subunit. We used biochemical and effector regulation data from human G protein studies to identify four potentially important residues for site-directed mutagenesis (T65, M111, D250 and W361 of AGB1). Each residue was individually mutated and the resulting mutated protein introduced in the agb1-2 mutant background under the control of the native AGB1 promoter. Interestingly, even though these mutations have been shown to have profound effects on effector signaling in humans, all the mutated subunits were able to restore thirteen of the fifteen Gβ-deficient phenotypes characterized in this study. Only one mutated protein, T65A was unable to complement the hypersensitivity to mannitol during germination observed in agb1 mutants; while only D250A failed to restore lateral root numbers in the agb1 mutant to wild-type levels. Our results suggest that the mechanisms used in mammalian G protein signaling are not well conserved in plant G protein signaling, and that either the effectors used by plant G proteins, or the mechanisms used to activate them, are at least partially divergent from the well-studied mammalian G proteins.

  17. Site-directed mutagenesis from Arg195 to His of a microalgal chloroplastidial glycerol-3-phosphate acyltransferase causes an increase in phospholipid levels in yeast

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    Long-Ling eOuyang

    2016-03-01

    Full Text Available To analyze the contribution of glycerol-3-phosphate acyltransferase (GPAT to the first acylation of glycerol-3-phosphate (G-3-P, the present study focused on a functional analysis of the GPAT gene from Lobosphaera incisa (designated as LiGPAT and the subcellular localization of the encoded protein LiGPAT. A full-length cDNA of LiGPAT consisting of a 1,305-bp ORF, a 1,652-bp 5′-UTR, and a 354-bp 3′-UTR, was cloned. The ORF encoded a 434-amino acid peptide, of which 63 residues at the N-terminus defined a chloroplast transit peptide. LiGPAT was exclusively localized to chloroplasts, which was shown by co-expression of LiGPAT with eGFP in Chlamydomonas reinhardtii and by immunogold labeling in L. incisa. Considering the conservation of His among the G-3-P binding sites from chloroplastidial GPATs and the substitution of His by Arg at position 195 in the LiGPAT mature protein (designated mLiGPAT, we established the heterologous expression of either mLiGPAT or its mutant (Arg195His (sdmLiGPAT in the GPAT-deficient yeast mutant gat1Δ. Lipid profile analyses of these transgenic yeasts not only validated the acylation function of LiGPAT but also indicated that the site-directed mutagenesis from Arg195 to His led to an increase in the phospholipid level in yeast. Semi-quantitative analysis of mLiGPAT and sdmLiGPAT, together with the structural superimposition of their G-3-P binding sites, indicated that the increased enzymatic activity was caused by the enlarged accessible surface of the phosphate group binding pocket when Arg195 was mutated to His. Thus, the potential of genetic manipulation of GPAT to increase the glycerolipid level in L. incisa and other microalgae would be of great interest.

  18. Site-directed mutagenesis of the active site of diacylglycerol kinase alpha: calcium and phosphatidylserine stimulate enzyme activity via distinct mechanisms.

    Science.gov (United States)

    Abe, Takahiro; Lu, Xiaolan; Jiang, Ying; Boccone, Clark E; Qian, Shaomin; Vattem, Krishna M; Wek, Ronald C; Walsh, James P

    2003-11-01

    Diacylglycerol kinases (DAGKs) catalyse ATP-dependent phosphorylation of sn-1,2-diacylglycerol that arises during stimulated phosphatidylinositol turnover. DAGKa is activated in vitro by Ca2+ and by acidic phospholipids. The regulatory region of DAGKa includes an N-terminal RVH motif and EF hands that mediate Ca2+-dependent activation. DAGKa also contains tandem C1 protein kinase C homology domains. We utilized yeast, Saccharomyces cerevisiae, which lacks an endogenous DAGK, to express DAGKa and to determine the enzymic activities of different mutant forms of pig DAGKa in vitro. Six aspartate residues conserved in all DAGKs were individually examined by site-directed mutagenesis. Five of these aspartate residues reside in conserved blocks that correspond to sequences in the catalytic site of phosphofructokinases. Mutation of D434 (Asp434) or D650 abolished all DAGKa activity, whereas substitution of one among D465, D497, D529 and D697 decreased the activity to 6% or less of that for wild-type DAGKa. Roles of homologous residues in phosphofructokinases suggested that the N-terminal half of the DAGK catalytic domain binds Mg-ATP and the C-terminal half binds diacylglycerol. A DAGKa mutant with its entire regulatory region deleted showed a much decreased activity that was not activated by Ca2+, but still exhibited PS (phosphatidylserine)-dependent activation. Moreover, mutations of aspartate residues at the catalytic domain had differential effects on activation by Ca2+ and PS. These results indicate that Ca2+ and PS stimulate DAGKa via distinct mechanisms.

  19. Site-directed mutagenesis of α-L-rhamnosidase from Alternaria sp. L1 to enhance synthesis yield of reverse hydrolysis based on rational design.

    Science.gov (United States)

    Xu, Li; Liu, Xiaohong; Yin, Zhenhao; Liu, Qian; Lu, Lili; Xiao, Min

    2016-12-01

    The α-L-rhamnosidase catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides and is widely used due to its applications in a variety of industrial processes. Our previous work reported that a wild-type α-L-rhamnosidase (RhaL1) from Alternaria sp. L1 could synthesize rhamnose-containing chemicals (RCCs) though reverse hydrolysis reaction with inexpensive rhamnose as glycosyl donor. To enhance the yield of reverse hydrolysis reaction and to determine the amino acid residues essential for the catalytic activity of RhaL1, site-directed mutagenesis of 11 residues was performed in this study. Through rationally designed mutations, the critical amino acid residues which may form direct or solvent-mediated hydrogen bonds with donor rhamnose (Asp(252), Asp(257), Asp(264), Glu(530), Arg(548), His(553), and Trp(555)) and may form the hydrophobic pocket in stabilizing donor (Trp(261), Tyr(302), Tyr(316), and Trp(369)) in active-site of RhaL1 were analyzed, and three positive mutants (W261Y, Y302F, and Y316F) with improved product yield stood out. From the three positive variants, mutant W261Y accelerated the reverse hydrolysis with a prominent increase (43.7 %) in relative yield compared to the wild-type enzyme. Based on the 3D structural modeling, we supposed that the improved yield of mutant W261Y is due to the adjustment of the spatial position of the putative catalytic acid residue Asp(257). Mutant W261Y also exhibited a shift in the pH-activity profile in hydrolysis reaction, indicating that introducing of a polar residue in the active site cavity may affect the catalysis behavior of the enzyme.

  20. Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    Science.gov (United States)

    Zheng, Zhong-liang; Ye, Mao-qing; Zuo, Zhen-yu; Liu, Zhi-gang; Tai, Keng-chang; Zou, Guo-lin

    2006-05-01

    Hydrogen bonds occurring in the catalytic triad (Asp32, His64 and Ser221) and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Ser33, Asp60, Ser62 and Thr220. In order to remove the effect of these hydrogen bonds, four mutants (Ser33-->Ala33, Asp60-->Ala60, Ser62-->Ala62, and Thr220-->Ala220) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (DeltaDeltaG(T)). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the kcat but not the K(m) values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Asp32, His64 and Asn155 so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-p-nitroanilide), was used for FEP calculations and MD simulations.

  1. Study of human Orexin-1 and -2 G-protein-coupled receptors with novel and published antagonists by modeling, molecular dynamics simulations, and site-directed mutagenesis.

    Science.gov (United States)

    Heifetz, Alexander; Morris, G Benjamin; Biggin, Philip C; Barker, Oliver; Fryatt, Tara; Bentley, Jonathan; Hallett, David; Manikowski, Dominique; Pal, Sandeep; Reifegerste, Rita; Slack, Mark; Law, Richard

    2012-04-17

    The class A G-protein-coupled receptors (GPCRs) Orexin-1 (OX1) and Orexin-2 (OX2) are located predominantly in the brain and are linked to a range of different physiological functions, including the control of feeding, energy metabolism, modulation of neuro-endocrine function, and regulation of the sleep-wake cycle. Site-directed mutagenesis (SDM) and domain exchange (chimera) studies have provided important insight into key features of the OX1 and OX2 binding sites. However, the precise determinants of antagonist binding and selectivity are still not fully known. In this work, we used homology modeling of OX receptors to direct further SDM studies. These SDM studies were followed by molecular dynamics (MD) simulations to rationalize the full scope of the SDM data and to explain the role of each mutated residue in the binding and selectivity of a set of OX antagonists: Almorexant (dual OX1 and OX2 antagonist), SB-674042 (OX1 selective antagonist), EMPA (OX2 selective antagonist), and others. Our primary interest was focused on transmembrane helix 3 (TM3), which is identified as being of great importance for the selectivity of OX antagonists. These studies revealed conformational differences between the TM3 helices of OX1 and OX2, resulting from differences in amino acid sequences of the OX receptors that affect key interhelical interactions formed between TM3 and neighboring TM domains. The MD simulation protocol used here, which was followed by flexible docking studies, went beyond the use of static models and allowed for a more detailed exploration of the OX structures. In this work, we have demonstrated how even small differences in the amino acid sequences of GPCRs can lead to significant differences in structure, antagonist binding affinity, and selectivity of these receptors. The MD simulations allowed refinement of the OX receptor models to a degree that was not possible with static homology modeling alone and provided a deeper rationalization of the SDM

  2. Signal transduction by the formyl peptide receptor. Studies using chimeric receptors and site-directed mutagenesis define a novel domain for interaction with G-proteins.

    Science.gov (United States)

    Amatruda, T T; Dragas-Graonic, S; Holmes, R; Perez, H D

    1995-11-24

    The binding of small peptide ligands to high affinity chemoattractant receptors on the surface of neutrophils and monocytes leads to activation of heterotrimeric G-proteins, stimulation of phosphatidylinositol-phospholipase C (PI-PLC), and subsequently to the inflammatory response. It was recently shown (Amatruda, T. T., Gerard, N. P., Gerard, C., and Simon, M. I. (1993) J. Biol. Chem. 268, 10139-10144) that the receptor for the chemoattractant peptide C5a specifically interacts with G alpha 16, a G-protein alpha subunit of the Gq class, to trigger ligand-dependent stimulation of PI-PLC in transfected cells. In order to further characterize this chemoattractant peptide signal transduction pathway, we transfected cDNAs encoding the formylmethionylleucylphenylalanine receptor (fMLPR) into COS cells and measured the production of inositol phosphates. Ligand-dependent activation of PI-PLC was seen in COS cells transfected with the fMLPR and G alpha 16 and stimulated with fMLP but not in cells transfected with receptor alone or with receptor plus G alpha q. Chimeric receptors in which the N-terminal extracellular domain, the second intracellular domain, or the intracellular C-terminal tail of the fMLP receptor was replaced with C5a receptor domains (Perez, H. D., Holmes, R., Vilander, L. R., Adams, R. R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295) were capable of ligand-dependent activation of PI-PLC when co-transfected with G alpha 16. A chimeric receptor exchanging the first intracellular domain of the fMLPR was constitutively activated, stimulating PI-PLC in the absence of ligand. Constitutive activation of PI-PLC, to a level 233% of that seen in cells transfected with wild-type fMLP receptors, was dependent on G alpha 16. Site-directed mutagenesis of the first intracellular domain of the fMLPR (amino acids 54-62) reveals this to be a domain necessary for ligand-dependent activation of G alpha 16. These results suggest that

  3. Modulation of the affinity of the single-stranded DNA-binding protein of Escherichia coli (E. coli SSB) to poly(dT) by site-directed mutagenesis.

    Science.gov (United States)

    Bayer, I; Fliess, A; Greipel, J; Urbanke, C; Maass, G

    1989-02-01

    A vector for site-directed mutagenesis and overproduction of the Escherichia coli single-stranded-DNA-binding protein (E. coli SSB) was constructed. An E. coli strain carrying this vector produces up to 400 mg pure protein from 25 g wet cells. The vector was used to mutate specifically the Phe60 residue of E. coli SSB. Phe60 had been proposed to be located near the single-stranded-DNA-binding site. Substitution of the Phe60 residue by Val, Ser, Leu, His, Tyr and Trp gave proteins with no or only minor conformational changes, as detected by NMR spectroscopy. The affinity of the mutant E. coli SSB proteins for single-stranded DNA decreased in the order Trp greater than Phe (wild-type) greater than Tyr greater than Leu greater than His greater than Val greater than Ser, leading to the conclusion that position 60 is a site of hydrophobic interaction of the protein with DNA.

  4. 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)

  5. Functional mapping of the fission yeast DNA polymerase δ B-subunit Cdc1 by site-directed and random pentapeptide insertion mutagenesis

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    Gray Fiona C

    2009-08-01

    Full Text Available Abstract Background DNA polymerase δ plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand. In fission yeast, Pol δ is a heterotetrameric enzyme comprising four evolutionarily well-conserved proteins: the catalytic subunit Pol3 and three smaller subunits Cdc1, Cdc27 and Cdm1. Pol3 binds directly to the B-subunit, Cdc1, which in turn binds the C-subunit, Cdc27. Human Pol δ comprises the same four subunits, and the crystal structure was recently reported of a complex of human p50 and the N-terminal domain of p66, the human orthologues of Cdc1 and Cdc27, respectively. Results To gain insights into the structure and function of Cdc1, random and directed mutagenesis techniques were used to create a collection of thirty alleles encoding mutant Cdc1 proteins. Each allele was tested for function in fission yeast and for binding of the altered protein to Pol3 and Cdc27 using the two-hybrid system. Additionally, the locations of the amino acid changes in each protein were mapped onto the three-dimensional structure of human p50. The results obtained from these studies identify amino acid residues and regions within the Cdc1 protein that are essential for interaction with Pol3 and Cdc27 and for in vivo function. Mutations specifically defective in Pol3-Cdc1 interactions allow the identification of a possible Pol3 binding surface on Cdc1. Conclusion In the absence of a three-dimensional structure of the entire Pol δ complex, the results of this study highlight regions in Cdc1 that are vital for protein function in vivo and provide valuable clues to possible protein-protein interaction surfaces on the Cdc1 protein that will be important targets for further study.

  6. Identification of essential residues for binding and activation in the human 5-HT7(a receptor by molecular modeling and site-directed mutagenesis

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    Agata Antonina Rita eImpellizzeri

    2015-05-01

    Full Text Available The human 5-HT7 receptor is expressed in both the central nervous system and peripheral tissues and is a potential drug target in behavioral and psychiatric disorders.We examined molecular determinants of ligand binding and G protein activation by the human 5-HT7(a receptor. The role of several key residues in the 7th transmembrane domain and helix 8 were elucidated combining in silico and experimental mutagenesis. Several single and two double point mutations of the 5-HT7(a wild type receptor were made (W7.33V, E7.35T, E7.35R, E7.35D, E7.35A, R7.36V, Y7.43A, Y7.43F, Y7.43T, R8.52D, D8.53K; E7.35T-R7.36V, R8.52D-D8.53K, and their effects upon ligand binding were assessed by radioligand binding using a potent agonist (5-CT and a potent antagonist (SB269970. In addition, the ability of the mutated 5-HT7(a receptors to activate G protein after 5-HT-stimulation was determined through activation of adenylyl cyclase. In silico investigation on mutated receptors substantiated the predicted importance of TM7 and showed critical roles of residues E7.35, W7.33, R7.36 and Y7.43 in agonist and antagonist binding and conformational changes of receptor structure affecting adenylyl cyclase activation. Experimental data showed that mutants E7.35T and E7.35R were incapable of ligand binding and adenylyl cyclase activation, consistent with a requirement for a negatively charged residue at this position. The mutant R8.52D was unable to activate adenylyl cyclase, despite unaffected ligand binding, consistent with the R8.52 residue playing an important role in the receptor-G protein interface. The mutants Y7.43A and Y7.43T displayed reduced agonist binding and AC agonist potency, not seen in Y7.43F, consistent with a requirement for an aromatic residue at this position. Knowledge of the molecular interactions important in h5-HT7 receptor ligand binding and G protein activation will aid the design of selective h5-HT7 receptor ligands for potential pharmacological use.

  7. 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.

  8. Site-directed mutagenesis of the -10 region of the lacUV5 promoter. Introduction of dA4.dT4 tract suppresses open complex formation.

    Science.gov (United States)

    Chan, P T; Lebowitz, J

    1990-03-05

    Homopolymeric dAn.dTn sequences, where n is 4 or greater, have special properties leading to increased duplex stability and DNA bending. The lacUV5 promoter was used to examine the functional consequences of changing the -10 TATAAT consensus sequence to the sequence TAAAAT. The transversion mutation at the underlined site was accomplished with site-directed mutagenesis using translation termination as the selection procedure. For free DNA, structural differences at the 5' and 3' junction regions of the dA4.dT4 tract can be readily detected by DNase I digestion. However, site binding by Escherichia coli RNA polymerase appeared unaltered by the TAAAAT sequence since identical DNase I footprints were obtained for the lacUV5 and mutant promoters. Binding competition studies under different ionic strengths revealed a significant reduction in mutant promoter open complex formation relative to the lacUV5 promoter. Mutant promoter open complexes also dissociated faster and to a greater extent than the corresponding lacUV5 promoter open complexes when challenged with heparin or a combination of heparin and increased KCl concentration. Consequently, mutant promoter open complexes appear less stable than lacUV5 promoter open complexes.

  9. Identification of the amino acid residue in the nematode galectin LEC-1 responsible for its unique sugar binding property: analysis by combination of site-directed mutagenesis and frontal affinity chromatography.

    Science.gov (United States)

    Arata, Yoichiro; Ishii, Norihide; Tamura, Mayumi; Nonaka, Takamasa; Kasai, Ken-ichi

    2007-11-01

    The basic disaccharide structure recognized by galectin family members is the lactosamine-like structure Galbeta1-4(3)Glc(NAc). In galectins, eight highly conserved amino acid residues participate in the recognition of this basic structure. Each galectin seems to mediate diverse biological functions due to recognition of different modifications of the basic disaccharide Galbeta1-4(3)Glc(NAc), but there is very little information about which amino acid residue in galectin is responsible for recognizing these modifications. The 32-kDa galectin LEC-1 of the nematode Caenorhabditis elegans is composed of two domains, each of which is homologous to vertebrate 14-kDa-type galectins. Although both lectin domains have an affinity for N-acetyllactosamine (Galbeta1-4GlcNAc)-containing, N-linked, complex-type sugar chains, the N-terminal lectin domain of LEC-1 recognizes blood group A saccharide (GalNAcalpha1-3(Fucalpha1-2)Galbeta1-3GlcNAc), whereas this saccharide is only poorly recognized by the C-terminal domain. Here, we used a combination of site-directed mutagenesis of the N-terminal lectin domain of galectin LEC-1 and an analysis of the sugar-binding profile by frontal affinity chromatography to identify the amino acid residues important for this recognition. Our results indicate that Thr(41) in the N-terminal lectin domain of LEC-1 is important for its affinity for A-hexasaccharide.

  10. 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.

  11. Proteoglycan: site mapping and site-directed mutagenesis.

    Science.gov (United States)

    Hagen, Fred K

    2012-01-01

    Identification of proteoglycan chain modification sites cannot yet be reliably predicted from primary amino acid sequence data. A number of studies have shown that serine is the predominant amino acid that is modified and it is frequently flanked by a C-terminal glycine and proximal N-terminal acidic amino acids; however, not all simple Ser-Gly motifs constitute a modification site. Here we present a rapid method for cloning small, defined segments of putative proteoglycan attachment sites and expressing them as a mini-reporter protein in an insect tissue culture system that is expandable to high throughput analysis. Reporter proteins with attached proteoglycans can be readily discerned from their unmodified form, by a simple gel-shift assay and Western blot detection for an epitope tag engineered into the reporter. Unmodified proteins are generated as a reference standard by treating cells with dsRNA to knock down the endogenous polypeptide xylose transferase, which is responsible for initiating proteoglycan site attachment. Examination of proteoglycan attachment by different metazoan organisms can be studied in the same cell line by cotransfecting a polypeptide xylose transferase expression plasmid and reporter construct from human, mouse, frog, or worm, for example. Reporter proteins engineer with point mutations can be rapidly generated with this system to pinpoint the exact residue that is glycosylated, to verify the mapping data.

  12. Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome

    Directory of Open Access Journals (Sweden)

    Berghman Luc R

    2007-09-01

    Full Text Available Abstract Background A variety of techniques have been described which introduce scarless, site-specific chromosomal mutations. These techniques can be applied to make point mutations or gene deletions as well as insert heterologous DNA into bacterial vectors for vaccine development. Most methods use a multi-step approach that requires cloning and/or designing repeat sequences to facilitate homologous recombination. We have modified previously published techniques to develop a simple, efficient PCR-based method for scarless insertion of DNA into Salmonella enteritidis chromosome. Results The final product of this mutation strategy is the insertion of DNA encoding a foreign epitope into the S. enteritidis genome without the addition of any unwanted sequence. This experiment was performed by a two-step mutation process via PCR fragments, Red recombinase and counter-selection with the I-SceI enzyme site. First, the I-SceI site and kanamycin resistance gene were introduced into the genome of cells expressing Red recombinase enzymes. Next, this sequence was replaced by a chosen insertion sequence. DNA fragments used for recombination were linear PCR products which consisted of the foreign insertion sequence flanked by homologous sequences of the target gene. Described herein is the insertion of a section of the M2e epitope (LM2 of Influenza A virus, a domain of CD154 (CD154s or a combination of both into the outer membrane protein LamB of S. enteritidis. Conclusion We have successfully used this method to produce multiple mutants with no antibiotic gene on the genome or extra sequence except those nucleotides required for expression of epitope regions. This method is advantageous over other protocols in that it does not require cloning or creating extra duplicate regions to facilitate homologous recombination, contains a universal construct in which an epitope of choice can be placed to check for cell surface expression, and shows high efficiency when screening for positive mutants. Other opportunities of this mutational strategy include creating attenuated mutants and site-specific, chromosomal deletion mutations. Furthermore, this method should be applicable in other gram-negative bacterial species where Red recombinase enzymes can be functionally expressed.

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

    Indian Academy of Sciences (India)

    2012-09-29

    Sep 29, 2012 ... ces, containing suitable restriction-enzyme-cutting sites, were added at the 5′ ends of primer F1 and R3 respectively, which provided combination sites for the full-length forward. (FF) primer and full-length reverse (FR) primer respectively. Low concentration of primers was used so that the primers could be ...

  14. Forward and reverse mutagenesis in C. elegans.

    Science.gov (United States)

    Kutscher, Lena M; Shaham, Shai

    2014-01-17

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

  15. Clustered regulatory interspaced short palindromic repeats (CRISPR)-mediated mutagenesis and phenotype rescue by piggyBac transgenesis in a nonmodel Drosophila species.

    Science.gov (United States)

    Tanaka, R; Murakami, H; Ote, M; Yamamoto, D

    2016-08-01

    How behavioural diversity emerged in evolution is an unexplored subject in biology. To tackle this problem, genes and circuits for a behaviour need to be determined in different species for phylogenetic comparisons. The recently developed clustered regulatory interspaced short palindromic repeats/CRISPR associated protein9 (CRISPR/Cas9) system made such a challenge possible by providing the means to induce mutations in a gene of interest in any organism. Aiming at elucidating diversification in genetic and neural networks for courtship behaviour, we attempted to generate a genetic tool kit in Drosophila subobscura, a nonmodel species distantly related to the genetic model Drosophila melanogaster. Here we report the generation of yellow (y) and white mutations with the aid of the CRISPR/Cas9 system, and the rescue of the y mutant phenotype by germline transformation of the newly established y mutant fly line with a y(+) -marked piggyBac vector. This successful mutagenesis and transformation in D. subobscura open up an avenue for comprehensive genetic analyses of higher functions in this and other nonmodel Drosophila species, representing a key step toward systematic comparisons of genes and circuitries underlying behaviour amongst species. © 2016 The Royal Entomological Society.

  16. Massively parallel single-amino-acid mutagenesis.

    Science.gov (United States)

    Kitzman, Jacob O; Starita, Lea M; Lo, Russell S; Fields, Stanley; Shendure, Jay

    2015-03-01

    Random mutagenesis methods only partially cover the mutational space and are constrained by DNA synthesis length limitations. Here we demonstrate programmed allelic series (PALS), a single-volume, site-directed mutagenesis approach using microarray-programmed oligonucleotides. We created libraries including nearly every missense mutation as singleton events for the yeast transcription factor Gal4 (99.9% coverage) and human tumor suppressor p53 (93.5%). PALS-based comprehensive missense mutational scans may aid structure-function studies, protein engineering, and the interpretation of variants identified by clinical sequencing.

  17. Massively Parallel Single Amino Acid Mutagenesis

    Science.gov (United States)

    Kitzman, Jacob O.; Starita, Lea M.; Lo, Russell S.; Fields, Stanley; Shendure, Jay

    2014-01-01

    Random mutagenesis methods only partially cover the mutational space, and are constrained by DNA synthesis length limitations. Here, we demonstrate PALS, a single-volume, site-directed mutagenesis approach using microarray-programmed oligonucleotides. We created libraries including nearly every missense mutation as singleton events for the yeast transcription factor Gal4 (99.9% coverage) and human tumor suppressor p53 (93.5%). PALS-based comprehensive missense mutational scans may aid structure-function studies, protein engineering, and the interpretation of variants identified by clinical sequencing. PMID:25559584

  18. Functional studies of elongation factor Tu from Escherichia coli : Site-directed mutagenesis and antibiotic action

    NARCIS (Netherlands)

    Krab, Ivo Maarten

    2001-01-01

    This PhD thesis describes several studies into the structure and function of Escherichia coli Elongation Factor Tu (EF-Tu). EF-Tu plays a central role in the bacterial protein synthesis machinery as the carrier of "coded building blocks" for protein synthesis, aminoacylated tRNA (aa-tRNA). Without

  19. Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor.

    Science.gov (United States)

    Deyev, Igor E; Chachina, Natalia A; Zhevlenev, Egor S; Petrenko, Alexander G

    2017-11-19

    The orphan insulin receptor-related receptor (IRR), in contrast to its close homologs, the insulin receptor (IR) and insulin-like growth factor receptor (IGF-IR) can be activated by mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by its extracellular region, involves multiple domains, and shows positive cooperativity with two synergistic sites. By the analyses of point mutants and chimeras of IRR with IR in, we now address the role of the fibronectin type III (FnIII) repeats in the IRR pH-sensing. The first activation site includes the intrinsically disordered subdomain ID (646-716) within the FnIII-2 domain at the C-terminus of IRR alpha subunit together with closely located residues L135, G188, R244, H318, and K319 of L1 and C domains of the second subunit. The second site involves residue T582 of FnIII-1 domain at the top of IRR lambda-shape pyramid together with M406, V407, and D408 from L2 domain within the second subunit. A possible importance of the IRR carbohydrate moiety for its activation was also assessed. IRR is normally less glycosylated than IR and IGF-IR. Swapping both FnIII-2 and FnIII-3 IRR domains with those of IR shifted beta-subunit mass from 68 kDa for IRR to about 100 kDa due to increased glycosylation and abolished the IRR pH response. However, mutations of four asparagine residues, potential glycosylation sites in chimera IRR with swapped FnIII-2/3 domains of IR, decreased the chimera glycosylation and resulted in a partial restoration of IRR pH-sensing activity, suggesting that the extensive glycosylation of FnIII-2/3 provides steric hindrance for the alkali-induced rearrangement of the IRR ectodomain.

  20. Catalytic efficiency and thermostability improvement of Suc2 invertase through rational site-directed mutagenesis.

    Science.gov (United States)

    Mohandesi, Nooshin; Haghbeen, Kamahldin; Ranaei, Omid; Arab, Seyed Shahriar; Hassani, Sorour

    2017-01-01

    Engineering of invertases has come to attention because of increasing demand for possible applications of invertases in various industrial processes. Due to the known physicochemical properties, invertases from micro-organisms such as Saccharomyces cerevisiae carrying SUC2 gene are considered as primary models. To improve thermostability and catalytic efficiency of SUC2 invertase (SInv), six influential residues with Relative Solvent Accessibilityinvertases with enhanced catalytic efficiency. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. 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.

  2. Site directed mutagenesis in Petunia using CRISP/CAS9 technology

    NARCIS (Netherlands)

    Dr. Nelleke Kreike; Paulus den Hollander

    2013-01-01

    The Green Biotechnology research group focusses on the application of molecular breeding/biotechnological tools and also on the development/analysis of new tools, for the breeding of enhanced vegetable crops and ornamental plants. The research group is positioned within Inholland University of

  3. Tweaking agonist efficacy at N-methyl-D-aspartate receptors by site-directed mutagenesis

    DEFF Research Database (Denmark)

    Hansen, Kasper B; Clausen, Rasmus P; Bjerrum, Esben J

    2005-01-01

    The structural basis for partial agonism at N-methyl-D-aspartate (NMDA) receptors is currently unresolved. We have characterized several partial agonists at the NR1/NR2B receptor and investigated the mechanisms underlying their reduced efficacy by introducing mutations in the glutamate binding si...

  4. Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II.

    Science.gov (United States)

    Verma, Shikha; Mehta, Ranjit Kumar; Maiti, Prasanta; Röhm, Klaus-Heinrich; Sonawane, Avinash

    2014-07-01

    Bacterial asparaginases (EC 3.5.1.1) have attracted considerable attention because enzymes of this group are used in the therapy of certain forms of leukemia. Class II asparaginase from Escherichia coli (EcA), a homotetramer with a mass of 138 kDa, is especially effective in cancer therapy. However, the therapeutic potential of EcA is impaired by the limited stability of the enzyme in vivo and by the induction of antibodies in the patients. In an attempt to modify the properties of EcA, several variants with amino acid replacements at subunit interfaces were constructed and characterized. Chemical and thermal denaturation analysis monitored by activity, fluorescence, circular dichroism, and differential scanning calorimetry showed that certain variants with exchanges that weaken dimer-dimer interactions exhibited complex denaturation profiles with active dimeric and/or inactive monomeric intermediates appearing at low denaturant concentrations. By contrast, other EcA variants showed considerably enhanced activity and stability as compared to the wild-type enzyme. Thus, even small changes at a subunit interface may markedly affect EcA stability without impairing its catalytic properties. Variants of this type may have a potential for use in the asparaginase therapy of leukemia. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Characterization of inhibitor binding to human kinesin spindle protein by site-directed mutagenesis.

    Science.gov (United States)

    Marshall, C Gary; Torrent, Maricel; Williams, Olusegun; Hamilton, Kelly A; Buser, Carolyn A

    2009-04-01

    A number of inhibitors of kinesin spindle protein (KSP) have been described, which are known from X-ray crystallography studies to bind to an induced fit pocket defined by the L5 loop. We describe the characterization of eight mutant forms of KSP in which six residues that line this pocket have been altered. Mutants were analyzed by measuring rates of enzyme catalysis, in the presence and absence of six KSP inhibitors of four diverse structural classes and of varied ATP-competition status. Our analysis was in agreement with the model of binding established by the structural studies and suggests that binding energy is well distributed across functional groups in these molecules. The majority of the mutants retained significant enzymatic activity while diminishing inhibitor binding, indicating potential for the development of drug resistance. These data provide detailed information on interactions between inhibitor and binding pocket at the functional group level and enable the development of novel KSP inhibitors.

  6. Construction of an Expression and Site-Directed Mutagenesis System of Haloalkane Dehalogenase in Escherichia coli

    NARCIS (Netherlands)

    Schanstra, J.P.; Rink, R.; Pries, F.; Janssen, D.B.

    1993-01-01

    Haloalkane dehalogenase from Xanthobacter autotrophicus was efficiently expressed in Escherichia coli BL21(DE3) and E. coli JM101. After introduction of restriction sites hy PCR the haloalkane dehalogenase gene (dhlA) was translationally fused behind the T7 (φ10), trc, and tac promoters. This

  7. 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 fac...

  8. Structure-activity investigation on laccases by computational and site directed mutagenesis studies

    OpenAIRE

    Delavari, Azar

    2016-01-01

    Laccases belong to multi copper oxidase enzyme family (EC 1.10.3.2). Their capacity to oxidíze a wide range of substrates makes them very attractive for the industry and are growing in importance for environmentally-friendly synthesis. Laccases have three different copper sites including, type 1 (T1), type 2 (T2) and type 3 (T3). The function of the T1 site is shuttling electrons from the substrate to the trinuclear copper cluster. During the catalytic cycle of laccase, four electrons are re...

  9. Molecular analysis of sialoside binding to sialoadhesin by NMR and site-directed mutagenesis.

    Science.gov (United States)

    Crocker, P R; Vinson, M; Kelm, S; Drickamer, K

    1999-07-15

    The molecular interactions between sialoadhesin and sialylated ligands have been investigated by using proton NMR. Addition of ligands to the 12 kDa N-terminal immunoglobulin-like domain of sialoadhesin result in resonance shifts in the protein spectrum that have been used to determine the affinities of sialoadhesin for several sialosides. The results indicate that alpha2, 3-sialyl-lactose and alpha2,6-sialyl-lactose bind respectively 2- and 1.5-fold more strongly than does alpha-methyl-N-acetylneuraminic acid (alpha-Me-NeuAc). The resonances corresponding to the methyl protons within the N-acetyl moiety of sialic acid undergo upfield shifting and broadening during titrations, reflecting an interaction of this group with Trp2 in sialoadhesin as observed in co-crystals of the terminal domain with bound ligand. This resonance shift was used to measure the affinities of mutant and wild-type forms of sialoadhesin in which the first three domains are fused to the Fc region of human IgG1. Substitution of Arg97 by alanine completely abrogated measurable interaction with alpha-Me-NeuAc, whereas a conservative substitution with lysine resulted in a 10-fold decrease in affinity. These results provide the first direct measurement of the affinity of sialoadhesin for sialosides and confirm the critical importance of the conserved arginine in interactions between sialosides and members of the siglec family of sialic acid-binding, immunoglobulin-like lectins.

  10. Functional analysis of truncated and site-directed mutagenesis dextransucrases to produce different type dextrans.

    Science.gov (United States)

    Wang, Chao; Zhang, Hong-Bin; Li, Meng-Qi; Hu, Xue-Qin; Li, Yao

    2017-07-01

    Dextrans with distinct molecular size and structure are increasingly being used in the food and pharmaceutical industries. Dextran is produced by dextransucrase (DSR, EC2.4.5.1), which is produced by Leuconostoc mesenteroides. DSR belongs to glycosyl hydrolase family (GH70) and synthesizes branched α-glucan (dextran) with both 5% α(1-3) and 95% α(1-6) glycosidic linkages. The DSR gene dex-YG (Genebank, Accession No. DQ345760) was cloned from the wild strain Leuconostoc mesenteroides 0326. This study generated a series of C-terminally truncated variants of dextransucrase and substituting the amino-acid residues in the active site of DSR. With shorter length of DSR, its polysaccharide-synthesizing capability was impaired heavily, whereas oligosaccharide (acting as prebiotics)-synthesizing capability increased significantly, efficiently producing special sizes of dextran. All truncated mutant enzymes were active. Results demonstrated that the catalytic domain dextransucrase was likely in 800 aa or less. Based on the three-dimensional structure model of dextransucrase built through homology modeling methods, the DSR and its mutants with the acceptor substrate of maltose and donor substrate of sucrose were studied by molecular-docking method. Substituting these amino-acid residues significantly affected enzyme activities. Compared with the wild-type dextran, mutant enzymes catalyzed the synthesis of a-glucan with 1-9% α(1-3) and 90-98% α(1-6) branching linkages. Some mutants introduced a small amount of α(1-4) linkages and α(1-2) linkages. This strategy can be effectively used for the rational protein design of dextransucrase. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Model building of a thermolysin-like protease by mutagenesis

    NARCIS (Netherlands)

    Frigerio, F; Margarit, [No Value; Nogarotto, R; Grandi, G; Vriend, G; Hardy, F; Veltman, OR; Venema, G; Eijsink, VGH

    The present study concerns the use of site-directed mutagenesis experiments to optimize a three-dimensional model of the neutral protease of Bacillus subtilis (NP-sub), An initial model of NP-sub was constructed using the crystal structures of the homologous neutral proteases of Bacillus

  12. Step-By-Step In Vitro Mutagenesis: Lessons From Fucose-Binding Lectin PA-IIL.

    Science.gov (United States)

    Mrázková, Jana; Malinovská, Lenka; Wimmerová, Michaela

    2017-01-01

    Site-directed mutagenesis is a powerful technique which is used to understand the basis of interactions between proteins and their binding partners, as well as to modify these interactions. Methods of rational design that are based on detailed knowledge of the structure of a protein of interest are often used for preliminary investigations of the possible outcomes which can result from the practical application of site-directed mutagenesis. Also, random mutagenesis can be used in tandem with site-directed mutagenesis for an examination of amino acid "hotspots."Lectins are sugar-binding proteins which, among other functions, mediate the recognition of host cells by a pathogen and its adhesion to the host cell surface. Hence, lectins and their binding properties are studied and engineered using site-directed mutagenesis.In this chapter, we describe a site-directed mutagenesis method used for investigating the sugar binding pattern of the PA-IIL lectin from the pathogenic bacterium Pseudomonas aeruginosa. Moreover, procedures for the production and purification of PA-IIL mutants are described, and several basic methods for characterizing the mutants are discussed.

  13. CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening.

    Science.gov (United States)

    Ito, Yasuhiro; Nishizawa-Yokoi, Ayako; Endo, Masaki; Mikami, Masafumi; Toki, Seiichi

    2015-11-06

    Site-directed mutagenesis using genetic approaches can provide a wealth of resources for crop breeding as well as for biological research. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 endonuclease (CRISPR/Cas9) system is a novel strategy used to induce mutations in a specific genome region; the system functions in a variety of organisms, including plants. Here, we report application of the CRISPR/Cas9 system to efficient mutagenesis of the tomato genome. In this study, we targeted the tomato RIN gene, which encodes a MADS-box transcription factor regulating fruit ripening. Three regions within the gene were targeted and mutations consisting either of a single base insertion or deletion of more than three bases were found at the Cas9 cleavage sites in T0 regenerated plants. The RIN-protein-defective mutants produced incomplete-ripening fruits in which red color pigmentation was significantly lower than that of wild type, while heterologous mutants expressing the remaining wild-type gene reached full-ripening red color, confirming the important role of RIN in ripening. Several mutations that were generated at three independent target sites were inherited in the T1 progeny, confirming the applicability of this mutagenesis system in tomato. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. 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.

  15. 2004 Mutagenesis Gordon Conference

    Energy Technology Data Exchange (ETDEWEB)

    Dr. Sue Jinks-Robertson

    2005-09-16

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

  16. Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides.

    Science.gov (United States)

    Rivera-Torres, Natalia; Banas, Kelly; Bialk, Pawel; Bloh, Kevin M; Kmiec, Eric B

    2017-01-01

    CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and

  17. Spectral tuning of photoproteins by partnering site-directed mutagenesis strategies with the incorporation of chromophore analogs.

    Science.gov (United States)

    Rowe, L; Rothert, A; Logue, C; Ensor, C M; Deo, S K; Daunert, S

    2008-02-01

    Aequorin and obelin are photoproteins whose calcium controlled bioluminescent light emission is used for labeling in assays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. Both of these photoproteins emit blue light from a 2-hydroperoxycoelenterazine chromophore, which is non-covalently bound in the hydrophobic core of the proteins. In an effort to produce aequorin and obelin variants with improved analytical properties, such as alternative emission colors and altered decay kinetics, seven mutants of aequorin and obelin were prepared and combined with 10 different coelenterazine analogs. These semi-synthetic photoprotein mutants exhibited shifts in bioluminescent properties when compared with wild-type proteins. The bioluminescent parameters determined for these semi-synthetic photoprotein mutants included specific activity, emission spectra and decay half-life time. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants that had significantly altered bioluminescent properties. The largest emission maxima shift obtained was 44 nm, and the largest decay half-life difference was 23.91 s.

  18. 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.

  19. Expansion of substrate specificity and catalytic mechanism of azoreductase by X-ray crystallography and site-directed mutagenesis.

    Science.gov (United States)

    Ito, Kosuke; Nakanishi, Masayuki; Lee, Woo-Cheol; Zhi, Yuehua; Sasaki, Hiroshi; Zenno, Shuhei; Saigo, Kaoru; Kitade, Yukio; Tanokura, Masaru

    2008-05-16

    AzoR is an FMN-dependent NADH-azoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. We previously reported the crystal structure of the enzyme in the oxidized form. In the present study, different structures of AzoR were determined under several conditions to obtain clues to the reaction mechanism of the enzyme. AzoR in its reduced form revealed a twisted butterfly bend of the isoalloxazine ring of the FMN cofactor and a rearrangement of solvent molecules. The crystal structure of oxidized AzoR in a different space group and the structure of the enzyme in complex with the inhibitor dicoumarol were also determined. These structures indicate that the formation of a hydrophobic part around the isoalloxazine ring is important for substrate binding and an electrostatic interaction between Arg-59 and the carboxyl group of the azo compound causes a substrate preference for methyl red over p-methyl red. The substitution of Arg-59 with Ala enhanced the Vmax value for p-methyl red 27-fold with a 3.8-fold increase of the Km value. This result indicates that Arg-59 decides the substrate specificity of AzoR. The Vmax value for the p-methyl red reduction of the R59A mutant is comparable with that for the methyl red reduction of the wild-type enzyme, whereas the activity toward methyl red was retained. These findings indicate the expansion of AzoR substrate specificity by a single amino acid substitution. Furthermore, we built an authentic model of the AzoR-methyl red complex based on the results of the study.

  20. Expansion of Substrate Specificity and Catalytic Mechanism of Azoreductase by X-ray Crystallography and Site-directed Mutagenesis

    National Research Council Canada - National Science Library

    Kosuke Ito; Masayuki Nakanishi; Woo-Cheol Lee; Yuehua Zhi; Hiroshi Sasaki; Shuhei Zenno; Kaoru Saigo; Yukio Kitade; Masaru Tanokura

    2008-01-01

    .... We previously reported the crystal structure of the enzyme in the oxidized form. In the present study, different structures of AzoR were determined under several conditions to obtain clues to the reaction mechanism of the enzyme...

  1. SITE-DIRECTED MUTAGENESIS OF PROPOSED ACTIVE-SITE RESIDUES OF PENICILLIN-BINDING PROTEIN-5 FROM ESCHERICHIA-COLI

    NARCIS (Netherlands)

    VANDERLINDEN, MPG; DEHAAN, L; DIDEBERG, O; KECK, W

    1994-01-01

    Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser(44)), Lys(47),

  2. 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...

  3. 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.

  4. Site-directed mutagenesis of an energy transducing protein: Bacteriorhodopsin. Final report, July 15, 1992--July 14, 1996

    Energy Technology Data Exchange (ETDEWEB)

    Needleman, R.

    1998-05-01

    The objective was to understand at the molecular level how bacteriorhodopsin (BR) transports protons. The work involves the synthesis of mutant BRs, their expression in the natural host, H. halobium, and an investigation of their photocycles. This final report has led to the development of a greatly improved expression system and to an increased understanding of the mechanism of proton transport. At the beginning of the award period a central concern was establishing the details of the photocycle. This phase was essentially complete by mid 1994. The author then investigated the energy coupling mechanism which allows uni-directional proton transfer and found that a major determinant was the coupling of the proton release to changes in the pKa of D85.

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

    Science.gov (United States)

    In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

  6. 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.

  7. Exploring the interaction of SV2A with racetams using homology modelling, molecular dynamics and site-directed mutagenesis.

    Directory of Open Access Journals (Sweden)

    Joanna Lee

    Full Text Available The putative Major Facilitator Superfamily (MFS transporter, SV2A, is the target for levetiracetam (LEV, which is a successful anti-epileptic drug. Furthermore, SV2A knock out mice display a severe seizure phenotype and die after a few weeks. Despite this, the mode of action of LEV is not known at the molecular level. It would be extremely desirable to understand this more fully in order to aid the design of improved anti-epileptic compounds. Since there is no structure for SV2A, homology modelling can provide insight into the ligand-binding site. However, it is not a trivial process to build such models, since SV2A has low sequence identity to those MFS transporters whose structures are known. A further level of complexity is added by the fact that it is not known which conformational state of the receptor LEV binds to, as multiple conformational states have been inferred by tomography and ligand binding assays or indeed, if binding is exclusive to a single state. Here, we explore models of both the inward and outward facing conformational states of SV2A (according to the alternating access mechanism for MFS transporters. We use a sequence conservation analysis to help guide the homology modelling process and generate the models, which we assess further with Molecular Dynamics (MD. By comparing the MD results in conjunction with docking and simulation of a LEV-analogue used in radioligand binding assays, we were able to suggest further residues that line the binding pocket. These were confirmed experimentally. In particular, mutation of D670 leads to a complete loss of binding. The results shed light on the way LEV analogues may interact with SV2A and may help with the on-going design of improved anti-epileptic compounds.

  8. Structural and biochemical characterization of two heme binding sites on α1-microglobulin using site directed mutagenesis and molecular simulation.

    Science.gov (United States)

    Rutardottir, Sigurbjörg; Karnaukhova, Elena; Nantasenamat, Chanin; Songtawee, Napat; Prachayasittikul, Virapong; Rajabi, Mohsen; Rosenlöf, Lena Wester; Alayash, Abdu I; Åkerström, Bo

    2016-01-01

    α1-Microglobulin (A1M) is a reductase and radical scavenger involved in physiological protection against oxidative damage. These functions were previously shown to be dependent upon cysteinyl-, C34, and lysyl side-chains, K(92, 118,130). A1M binds heme and the crystal structure suggests that C34 and H123 participate in a heme binding site. We have investigated the involvement of these five residues in the interactions with heme. Four A1M-variants were expressed: with cysteine to serine substitution in position 34, lysine to threonine substitutions in positions (92, 118, 130), histidine to serine substitution in position 123 and a wt without mutations. Heme binding was investigated by tryptophan fluorescence quenching, UV-Vis spectrophotometry, circular dichroism, SPR, electrophoretic migration shift, gel filtration, catalase-like activity and molecular simulation. All A1M-variants bound to heme. Mutations in C34, H123 or K(92, 118, 130) resulted in significant absorbance changes, CD spectral changes, and catalase-like activity, suggesting involvement of these side-groups in coordination of the heme-iron. Molecular simulation support a model with two heme-binding sites in A1M involving the mutated residues. Binding of the first heme induces allosteric stabilization of the structure predisposing for a better fit of the second heme. The results suggest that one heme-binding site is located in the lipocalin pocket and a second binding site between loops 1 and 4. Reactions with the hemes involve the side-groups of C34, K(92, 118, 130) and H123. The model provides a structural basis for the functional activities of A1M: heme binding activity of A1M. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. 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-up by ...... insertions per cell. MO-MAGE enables cost-effective large-scale targeted genome engineering that should be useful for a variety of applications in synthetic biology and metabolic engineering.......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...

  10. 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

  11. Technological advances in site-directed spin labeling of proteins.

    Science.gov (United States)

    Hubbell, Wayne L; López, Carlos J; Altenbach, Christian; Yang, Zhongyu

    2013-10-01

    Molecular flexibility over a wide time range is of central importance to the function of many proteins, both soluble and membrane. Revealing the modes of flexibility, their amplitudes, and time scales under physiological conditions is the challenge for spectroscopic methods, one of which is site-directed spin labeling EPR (SDSL-EPR). Here we provide an overview of some recent technological advances in SDSL-EPR related to investigation of structure, structural heterogeneity, and dynamics of proteins. These include new classes of spin labels, advances in measurement of long range distances and distance distributions, methods for identifying backbone and conformational fluctuations, and new strategies for determining the kinetics of protein motion. Copyright © 2013 Elsevier Ltd. All rights reserved.

  12. Ethyl methanesulfonate mutagenesis in Schizosaccharomyces pombe

    DEFF Research Database (Denmark)

    Ekwall, Karl; Thon, Genevieve

    2017-01-01

    Here we provide an ethyl methanesulfonate (EMS) mutagenesis protocol for Schizosaccharomyces pombe cells.......Here we provide an ethyl methanesulfonate (EMS) mutagenesis protocol for Schizosaccharomyces pombe cells....

  13. 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.

  14. 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

  15. Short (

    NARCIS (Netherlands)

    Telleman, Gerdien; den Hartog, Laurens

    2013-01-01

    Aim: This systematic review assessed the implant survival rate of short (<10 mm) dental implants installed in partially edentulous patients. A case report of a short implant in the posterior region have been added. Materials and methods: A search was conducted in the electronic databases of MEDLINE

  16. 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

    2017-05-16

    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.

  17. Promoter analysis by saturation mutagenesis

    Directory of Open Access Journals (Sweden)

    Baliga Nitin

    2001-01-01

    Full Text Available Gene expression and regulation are mediated by DNA sequences, in most instances, directly upstream to the coding sequences by recruiting transcription factors, regulators, and a RNA polymerase in a spatially defined fashion. Few nucleotides within a promoter make contact with the bound proteins. The minimal set of nucleotides that can recruit a protein factor is called a cis-acting element. This article addresses a powerful mutagenesis strategy that can be employed to define cis-acting elements at a molecular level. Technical details including primer design, saturation mutagenesis, construction of promoter libraries, phenotypic analysis, data analysis, and interpretation are discussed.

  18. 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.

  19. Site-Directed Research and Development FY 2014 Annual Report

    Energy Technology Data Exchange (ETDEWEB)

    Bender, H. A. [Nevada Test Site/National Security Technologies, LLC (United States)

    2015-04-22

    The reports contained herein are for project activities that occurred from October 2013 through September 2014. Project life cycle is indicated under the title as well as the original proposal number (in the following format: site abbreviation--ID #--originating fiscal year; e.g., STL-03-14). Each of the reports describes in detail the discoveries, achievements, and challenges encountered by our principal investigators. As SDRD, by definition, invests in “high-risk” and hopefully “high-payoff” research, the element of uncertainty is inherent. While many of our efforts are “successful” and result in positive outcomes or technology utilization, some fall short of expectations, but cannot be construed as “failure” in the negative sense. The latter is a natural and valid part of the process of advanced research and often leads to unforeseen new pathways to future discovery. Regardless, either result advances our knowledge base and increases our ability to identify solutions and/or avoid costly and unwarranted paths for future challenges. In summary, the SDRD program continues to provide an unfettered mechanism for innovation that returns multifold to our customers, to national security, and to the general public. The program is a vibrant R&D innovation engine, benefited by its discretionary pedigree, enhanced mission spectrum, committed resources, and sound competitiveness to yield maximum taxpayer benefit. The 25 projects described exemplify the creativity and ability of a diverse scientific and engineering talent base. The efforts also showcase an impressive capability and resource that can be brought to find solutions to a broad array of technology needs and applications relevant to the NNSS mission and national security. Further SDRD performance metrics can be found in the appendix at the end of this report.

  20. 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.

  1. Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA

    National Research Council Canada - National Science Library

    Svitashev, Sergei; Young, Joshua K; Schwartz, Christine; Gao, Huirong; Falco, S Carl; Cigan, A Mark

    2015-01-01

    Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas...

  2. 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...

  3. 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.

  4. 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

  5. Stabilization of Penicillin G Acylase from Escherichia coli: Site-Directed Mutagenesis of the Protein Surface To Increase Multipoint Covalent Attachment

    Science.gov (United States)

    Abian, Olga; Grazú, Valeria; Hermoso, Juan; González, Ramón; García, José Luis; Fernández-Lafuente, Roberto; Guisán, José Manuel

    2004-01-01

    Three mutations on the penicillin acylase surface (increasing the number of Lys in a defined area) were performed. They did not alter the enzyme's stability and kinetic properties; however, after immobilization on glyoxyl-agarose, the mutant enzyme showed improved stability under all tested conditions (e.g., pH 2.5 at 4°C, pH 5 at 60°C, pH 7 at 55°C, or 60% dimethylformamide), with stabilization factors ranging from 4 to 11 compared with the native enzyme immobilized on glyoxyl-agarose. PMID:14766616

  6. Mechanism of adenylate kinase. Demonstration of a functional relationship between aspartate 93 and Mg2+ by site-directed mutagenesis and proton, phosphorus-31, and magnesium-25 NMR.

    Science.gov (United States)

    Yan, H G; Tsai, M D

    1991-06-04

    Earlier magnetic resonance studies suggested no direct interaction between Mg2+ ions and adenylate kinase (AK) in the AK.MgATP (adenosine 5'-triphosphate) complex. However, recent NMR studies concluded that the carboxylate of aspartate 119 accepts a hydrogen bond from a water ligand of the bound Mg2+ ion in the muscle AK.MgATP complex [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. On the other hand, in the 2.6-A crystal structure of the yeast AK.MgAP5A [P1,P5-bis(5'-adenosyl)pentaphosphate] complex, the Mg2+ ion is in proximity to aspartate 93 [Egner, U., Tomasselli, A.G., & Schulz, G.E. (1987) J. Mol. Biol. 195, 649-658]. Substitution of Asp-93 with alanine resulted in no change in dissociation constants, 4-fold increases in Km, and a 650-fold decrease in kcat. Notable changes have been observed in the chemical shifts of the aromatic protons of histidine 36 and a few other aromatic residues. However, the results of detailed analyses of the free enzymes and the AK.MgAP5A complexes by one- and two-dimensional NMR suggested that the changes are due to localized perturbations. Thus it is concluded that Asp-93 stabilizes the transition state by ca. 3.9 kcal/mol. The next question is how. Since proton NMR results indicated that binding of Mg2+ to the AK.AP5A complex induces some changes in the proton NMR signals of WT but not those of D93A, the functional role of Asp-93 should be in binding to Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. SITE-DIRECTED MUTAGENESIS AND X-RAY CRYSTALLOGRAPHY OF 2 PHOSPHOLIPASE-A2 MUTANTS - Y52F AND Y73F

    NARCIS (Netherlands)

    THUNNISSEN, MMGM; FRANKEN, PA; DEHAAS, GH; DRENTH, J; KALK, KH; VERHEIJ, HM

    1992-01-01

    Tyr52 and Tyr73 are conserved amino acid residues throughout all vertebrate phospholipases A2. They are part of an extended hydrogen bonding system that links the N-terminal alpha-NH+-group to the catalytic residues His48 and Asp99. These tyrosines were replaced by phenylalanines in a porcine

  8. Mapping part of the functional epitope for ligand binding on the receptor for urokinase-type plasminogen activator by site-directed mutagenesis

    DEFF Research Database (Denmark)

    Gårdsvoll, H; Danø, K; Ploug, M

    1999-01-01

    ), Leu(55), Tyr(57), and Leu(66)). The energetic impact of these four alanine substitutions was not caused by gross structural perturbations, since all monoclonal antibodies tested having conformation-dependent epitopes on this domain exhibited unaltered binding kinetics. These sites together...... with a three-dimensional structure for uPAR may provide an appropriate target for rational drug design aimed at developing new receptor binding antagonists with potential application in cancer therapy....

  9. 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 of th...

  10. Identification of in vivo brain-derived neurotrophic factor-stimulated autophosphorylation sites on the TrkB receptor tyrosine kinase by site-directed mutagenesis.

    Science.gov (United States)

    Guiton, M; Gunn-Moore, F J; Stitt, T N; Yancopoulos, G D; Tavaré, J M

    1994-12-02

    Brain-derived neurotrophic factor (BDNF) interacts with the TrkB receptor tyrosine kinase, the tyrosine kinase domain of which has homology with the insulin receptor subfamily of protein kinases. This includes the conservation of three regulatory tyrosines (residues 670, 674, and 675) known to play a crucial role in signal transmission by the insulin receptor (tyrosines 1158, 1162, and 1163). Wild-type TrkB and TrkB mutants with Y670F, Y674F/Y675F, Y751F (the tyrosine reported to be important in phosphatidylinositol 3-kinase binding (Obermeier, A., Lammers, R., Wiesmuller, K. H., June, G., Schlessinger, J., and Ullrich, A. (1993) J. Biol. Chem. 268, 22963-22966)), and K540R (consensus ATP binding lysine) substitutions were transiently expressed in COS cells for analysis of phosphorylation sites by two-dimensional phosphopeptide mapping. TrkB phosphorylation sites were also studied in MG86 cells stably expressing wild-type TrkB. In addition, the mutants were expressed in Chinese hamster ovary cells for analysis of the ability of the receptor to mediate BDNF-stimulated transcription from a 12-O-tetradecanoylphorbol-13-acetate response element (TRE). BDNF stimulated the phosphorylation of wild-type TrkB on multiple tyrosine and serine residues. This phosphorylation occurred on tyrosines 670, 674, and 675 plus two other tyrosines and at least two serines that were not unequivocally identified. Wild-type TrkB mediated a pronounced stimulation of TRE-dependent transcription. A Y674F/Y675F, but not Y670F, substitution dramatically inhibited this response. Surprisingly, in COS cells, a Y751F substitution induced dramatically lower tyrosine and serine phosphorylation at all sites but mediated a normal BDNF-stimulated activation of a TRE. Our results demonstrate a critical role for the phosphorylation of tyrosines 674 and 675 in BDNF-dependent signaling by wild-type TrkB.

  11. 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,

  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 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 ...

  14. 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...

  15. Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase.

    OpenAIRE

    Mizrahi, V; Usdin, M T; Harington, A; Dudding, L R

    1990-01-01

    Substitution of the conserved Asp-443 residue of HIV-1 reverse transcriptase by asparagine specifically suppressed the ribonuclease H activity of the enzyme without affecting the reverse transcriptase activity, suggesting involvement of this ionizable residue at the ribonuclease H active site. An analogous asparagine substitution of the Asp-498 residue yielded an unstable enzyme that was difficult to enzymatically characterize. However, the instability caused by the Asn-498 mutation was relie...

  16. 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…

  17. 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.

  18. 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...

  19. Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS by Site-Directed Mutagenesis Improves Its Activity.

    Directory of Open Access Journals (Sweden)

    Aparajita Banerjee

    Full Text Available Deoxyxylulose 5-phosphate synthase (DXS, a thiamine diphosphate (ThDP dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP pathway. Isopentenyl diphosphate (IDP and dimethylallyl diphosphate (DMADP, the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity.

  20. Insights into the key interactions between human protein phosphatase 5 and cantharidin using molecular dynamics and site-directed mutagenesis bioassays

    OpenAIRE

    Ji-Yuan Liu; Xi-En Chen; Ya-Lin Zhang

    2015-01-01

    Serine/threonine protein phosphatase 5 (PP5) is a promising novel target for anticancer therapies. This work aims to uncover the key interactions at the atomic level between PP5 and three inhibitors (cantharidin, norcantharidin and endothall). We found that, unlike previous report, Arg 100 contributes less to PP5-inhibitor binding, and the residues His 69, Asn 128, His 129, Arg 225, His 252 and Arg 250 are of importance to PP5-inhibitor binding. The hydrophobic interactions established betwee...

  1. Insights into the key interactions between human protein phosphatase 5 and cantharidin using molecular dynamics and site-directed mutagenesis bioassays.

    Science.gov (United States)

    Liu, Ji-Yuan; Chen, Xi-En; Zhang, Ya-Lin

    2015-07-20

    Serine/threonine protein phosphatase 5 (PP5) is a promising novel target for anticancer therapies. This work aims to uncover the key interactions at the atomic level between PP5 and three inhibitors (cantharidin, norcantharidin and endothall). We found that, unlike previous report, Arg 100 contributes less to PP5-inhibitor binding, and the residues His 69, Asn 128, His 129, Arg 225, His 252 and Arg 250 are of importance to PP5-inhibitor binding. The hydrophobic interactions established between the residues Val 254, Phe 271 and Tyr 276, especially Glu 253, are very important to enhance the inhibitive interaction. We suggested that, to increase the inhibitory activity, the interactions of inhibitor with three negatively charged unfavorable interaction residues, Asp 99, Glu 130 and Asp 213, should be avoided. However, the interactions of inhibitor with favorable interaction residue Arg 250 could enhance the inhibitory activity. The Manganese ion 2 (MN2) unfavorably contribute to the total interaction free energies. The coordination between MN2 and chemical group of inhibitor should be eliminated. This work provides insight into how cantharidin and its analogs bind to PP5c at the atomic level and will facilitate modification of cantharidin-like chemicals to rationally develop more specific and less cytotoxic anti-cancer drugs.

  2. 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...

  3. Controlled Site-Directed Assembly of Antibodies by Their Oligosaccharide Moieties onto APTES Derivatized Surfaces.

    Science.gov (United States)

    Weiping; Bin; Lei; Chunxiao; Danfeng; Fang; Chunwei; Yu

    1999-06-01

    A convenient and efficient method for the site-directed incorporation of aldehydes generated on the oligosaccharide moieties at the C-terminal of immunoglobulin (IgG) using NaIO4 oxidation reaction is explored as a means of ensuring controlled assembly of IgG antibodies onto aminopropyltriethoxylsilane (APTES) derivatized silicon wafer surfaces. The orientation and antigen binding capacity (AgBC) of site-directly assembled IgG antibodies on derivatized surfaces were investigated using atomic force microscopy (AFM) and enzyme immunoassay (EIA), respectively. A major difference in preferential orientation is observed when the incubation of derivatized surfaces with oxidized IgG molecules is compared in two different kinds of buffer solutions. We obtained the stable and homogeneous IgG layer without loss of the AgBC on the APTES derivatized surface using the controlled incubation condition. Copyright 1999 Academic Press.

  4. Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries.

    Science.gov (United States)

    Huang, Renhua; Fang, Pete; Kay, Brian K

    2012-09-01

    Site-directed mutagenesis is routinely performed in protein engineering experiments. One method, termed Kunkel mutagenesis, is frequently used for constructing libraries of peptide or protein variants in M13 bacteriophage, followed by affinity selection of phage particles. To make this method more efficient, the following two modifications were introduced: culture was incubated at 25°C for phage replication, which yielded two- to sevenfold more single-stranded DNA template compared to growth at 37°C, and restriction endonuclease recognition sites were used to remove non-recombinants. With both of the improvements, we could construct primary libraries of high complexity and that were 99-100% recombinant. Finally, with a third modification to the standard protocol of Kunkel mutagenesis, two secondary (mutagenic) libraries of a fibronectin type III (FN3) monobody were constructed with DNA segments that were amplified by error-prone and asymmetric PCR. Two advantages of this modification are that it bypasses the lengthy steps of restriction enzyme digestion and ligation, and that the pool of phage clones, recovered after affinity selection, can be used directly to generate a secondary library. Screening one of the two mutagenic libraries yielded variants that bound two- to fourfold tighter to human Pak1 kinase than the starting clone. The protocols described in this study should accelerate the discovery of phage-displayed recombinant affinity reagents. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. 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.

  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. Controllable construction of carbohydrate microarrays by site-directed grafting on self-organized porous films.

    Science.gov (United States)

    Ke, Bei-Bei; Wan, Ling-Shu; Xu, Zhi-Kang

    2010-06-01

    Carbohydrate-protein interactions are critical in many biological processes. However, the interactions between individual carbohydrates and proteins are often of low affinity and difficult to study. Recent development of carbohydrate microarrays provides an effective tool to explore the interaction. In this work, carbohydrate microarrays were controllably constructed by grafting of a carbohydrate-containing monomer on self-organized honeycomb-patterned films. The films were prepared from an amphiphilic block copolymer, poly(styrene-block-(2-hydroxyethyl methacrylate)), by a breath figure method. Three-dimensional fluorescence results demonstrate that the hydroxyl groups aggregate mainly inside the pores, which afford a chance of site-directed surface modification. 2-(2,3,4,6-Tetra-O-acetyl-beta-D-glucosyloxy)ethyl methacrylate was selectively grafted in the pores by a surface-initiated atom transfer radical polymerization. Characterization by attenuated total reflectance Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy, and contact angle measurements confirms the site-directed growth of the glycopolymer chains. Further specific recognition of the carbohydrate microarrays to lectin (concanavalin A) leads to an organized microarray of protein, and hence this approach also opens a new route to fabricating other functional microarrays such as protein-patterned surfaces.

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

    DEFF Research Database (Denmark)

    Nielsen, Ulrik Dam

    2006-01-01

    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......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...... 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...

  9. Nonbinding site-directed mutants of transferrin binding protein B exhibit enhanced immunogenicity and protective capabilities.

    Science.gov (United States)

    Frandoloso, Rafael; Martínez-Martínez, Sonia; Calmettes, Charles; Fegan, Jamie; Costa, Estela; Curran, Dave; Yu, Rong-Hua; Gutiérrez-Martín, César B; Rodríguez-Ferri, Elías F; Moraes, Trevor F; Schryvers, Anthony B

    2015-03-01

    Host-adapted Gram-negative bacterial pathogens from the Pasteurellaceae, Neisseriaceae, and Moraxellaceae families normally reside in the upper respiratory or genitourinary tracts of their hosts and rely on utilizing iron from host transferrin (Tf) for growth and survival. The surface receptor proteins that mediate this critical iron acquisition pathway have been proposed as ideal vaccine targets due to the critical role that they play in survival and disease pathogenesis in vivo. In particular, the surface lipoprotein component of the receptor, Tf binding protein B (TbpB), had received considerable attention as a potential antigen for vaccines in humans and food production animals but this has not translated into the series of successful vaccine products originally envisioned. Preliminary immunization experiments suggesting that host Tf could interfere with development of the immune response prompted us to directly address this question with site-directed mutant proteins defective in binding Tf. Site-directed mutants with dramatically reduced binding of porcine transferrin and nearly identical structure to the native proteins were prepared. A mutant Haemophilus parasuis TbpB was shown to induce an enhanced B-cell and T-cell response in pigs relative to native TbpB and provide superior protection from infection than the native TbpB or a commercial vaccine product. The results indicate that binding of host transferrin modulates the development of the immune response against TbpBs and that strategies designed to reduce or eliminate binding can be used to generate superior antigens for vaccines. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

    Science.gov (United States)

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

    2016-04-01

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

  11. 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.

  12. Electron capture dissociation and drift tube ion mobility-mass spectrometry coupled with site directed mutations provide insights into the conformational diversity of a metamorphic protein.

    Science.gov (United States)

    Harvey, Sophie R; Porrini, Massimiliano; Tyler, Robert C; MacPhee, Cait E; Volkman, Brian F; Barran, Perdita E

    2015-04-28

    Ion mobility mass spectrometry can be combined with data from top-down sequencing to discern adopted conformations of proteins in the absence of solvent. This multi-technique approach has particular applicability for conformationally dynamic systems. Previously, we demonstrated the use of drift tube ion mobility-mass spectrometry (DT IM-MS) and electron capture dissociation (ECD) to study the metamorphic protein lymphotactin (Ltn). Ltn exists in equilibrium between distinct monomeric (Ltn10) and dimeric (Ltn40) folds, both of which can be preserved and probed in the gas-phase. Here, we further test this mass spectrometric framework, by examining two site directed mutants of Ltn, designed to stabilise either distinct fold in solution, in addition to a truncated form consisting of a minimum model of structure for Ltn10. The truncated mutant has similar collision cross sections to the wild type (WT), for low charge states, and is resistant to ECD fragmentation. The monomer mutant (CC3) presents in similar conformational families as observed previously for the WT Ltn monomer. As with the WT, the CC3 mutant is resistant to ECD fragmentation at low charge states. The dimer mutant W55D is found here to exist as both a monomer and dimer. As a monomer W55D exhibits similar behaviour to the WT, but as a dimer presents a much larger charge state and collision cross section range than the WT dimer, suggesting a smaller interaction interface. In addition, ECD on the W55D mutant yields greater fragmentation than for the WT, suggesting a less stable β-sheet core. The results highlight the power of MS to provide insight into dynamic proteins, providing further information on each distinct fold of Ltn. In addition we observe differences in the fold stability following single or double point mutations. This approach, therefore, has potential to be a useful tool to screen for the structural effects of mutagenesis, even when sample is limited.

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

    Science.gov (United States)

    Liu, Ting-ting; Fan, Di; Ran, Ling-yu; Jiang, Yuan-zhong; Liu, Rui; Luo, Ke-ming

    2015-10-01

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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Site-directed mutation of a laccase from Thermus thermophilus: Effect on the activity profile

    Directory of Open Access Journals (Sweden)

    Liu Xin

    2012-01-01

    Full Text Available A site-directed mutant R453T of a laccase from Thermus thermophilus HB27 (Tth-laccase was constructed in order to investigate the effect on laccase catalytic properties. The mutated gene was cloned and overexpressed in Escherichia coli. Nickel-affinity purification was achieved and followed by copper ion incorporation. The mature mutated enzyme was quantitatively equal to the wild type. A photometric assay based on the oxidation of the substrate 2,2-azino-bis-(3- ethylbenzthiazoline-6-sulfonate (ABTS was employed in comparison with the wild-type Tth-laccase on catalytic properties. The R453T mutant exhibited improvement in substrate affinity and specific activity at room temperature, whereas those parameters were not significantly influenced when the temperature increased up to 65°C or higher. The mutant had better catalytic activity than that of the wild type at acidic pH. Investigated by circular dichroism spectroscopy, the mutant Tth-laccase displayed similar profiles at low and high temperatures.

  19. Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice.

    Science.gov (United States)

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2016-05-01

    Recent reports of CRISPR- (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) mediated heritable mutagenesis in plants highlight the need for accuracy of the mutagenesis directed by this system. Off-target mutations are an important issue when considering functional gene analysis, as well as the molecular breeding of crop plants with large genome size, i.e. with many duplicated genes, and where the whole-genome sequence is still lacking. In mammals, off-target mutations can be suppressed by using Cas9 paired nickases together with paired guide RNAs (gRNAs). However, the performance of Cas9 paired nickases has not yet been fully assessed in plants. Here, we analyzed on- and off-target mutation frequency in rice calli and regenerated plants using Cas9 nuclease or Cas9 nickase with paired gRNAs. When Cas9 paired nickases were used, off-target mutations were fully suppressed in rice calli and regenerated plants. However, on-target mutation frequency also decreased compared with that induced by the Cas9 paired nucleases system. Since the gRNA sequence determines specific binding of Cas9 protein-gRNA ribonucleoproteins at the targeted sequence, the on-target mutation frequency of Cas9 paired nickases depends on the design of paired gRNAs. Our results suggest that a combination of gRNAs that can induce mutations at high efficiency with Cas9 nuclease should be used together with Cas9 nickase. Furthermore, we confirmed that a combination of gRNAs containing a one nucleotide (1 nt) mismatch toward the target sequence could not induce mutations when expressed with Cas9 nickase. Our results clearly show the effectiveness of Cas9 paired nickases in delivering on-target specific mutations. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  20. Long-distance effects of insertional mutagenesis.

    Directory of Open Access Journals (Sweden)

    Ruchi Singhal

    2011-01-01

    Full Text Available Most common systems of genetic engineering of mammalian cells are associated with insertional mutagenesis of the modified cells. Insertional mutagenesis is also a popular approach to generate random alterations for gene discovery projects. A better understanding of the interaction of the structural elements within an insertional mutagen and the ability of such elements to influence host genes at various distances away from the insertion site is a matter of considerable practical importance.We observed that, in the context of a lentiviral construct, a transcript, which is initiated at an internal CMV promoter/enhancer region and incorporates a splice donor site, is able to extend past a collinear viral LTR and trap exons of host genes, while the polyadenylation signal, which is naturally present in the LTR, is spliced out. Unexpectedly, when a vector, which utilizes this phenomenon, was used to produce mutants with elevated activity of NF-κB, we found mutants, which owed their phenotype to the effect of the insert on a gene located tens or even hundreds of kilobases away from the insertion site. This effect did not result from a CMV-driven transcript, but was sensitive to functional suppression of the insert. Interestingly, despite the long-distance effect, expression of loci most closely positioned to the insert appeared unaffected.We concluded that a polyadenylation signal in a retroviral LTR, when occurring within an intron, is an inefficient barrier against the formation of a hybrid transcript, and that a vector containing a strong enhancer may selectively affect the function of genes far away from its insertion site. These phenomena have to be considered when experimental or therapeutic transduction is performed. In particular, the long-distance effects of insertional mutagenesis bring into question the relevance of the lists of disease-associated retroviral integration targets, which did not undergo functional validation.

  1. Perspective on mutagenesis and repair: the standard model and alternate modes of mutagenesis.

    Science.gov (United States)

    Miller, Jeffrey H

    2005-01-01

    The basic ideas of replication, mutagenesis, and repair have outlined a picture of how point mutations occur that has provided a valuable framework for theory and experiment, much as the Standard Model of particle physics has done for our concept of fundamental particles. However, alternative modes of mutagenesis are being defined that are changing our perspective of the "Standard Model" of mutagenesis, requiring an expanded model. The genome is now envisioned as being in dynamic equilibrium between a multitude of forces for mutational change and forces that counteract such change. By maintaining a delicate balance between these forces, cells avoid unwanted or excessive mutations. Yet, cells allow mutagenesis to occur under certain conditions. We can define an emerging paradigm. Namely, mechanisms exist that can direct point mutations to specific designated genes or regions of genes. In some cases, this is achieved by specific enzymes, and in other cases high mutability is programmed into the sequence of certain genes to help generate diversity. In yet additional cases, general mutability is increased under stress, and selective forces allow the recovery of favorable mutants.

  2. 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.

  3. Purification of Functional CB1 and Analysis by Site-Directed Fluorescence Labeling Methods.

    Science.gov (United States)

    Fay, Jonathan F; Farrens, David L

    2017-01-01

    The human cannabinoid receptor, CB1, has been difficult to purify in a functional form, hampering structural and biophysical studies. Here, we present our approaches for obtaining pure, detergent solubilized, functional CB1. We also discuss our site-directed fluorescence labeling (SDFL) methods for identifying different structural changes that CB1 can undergo upon binding different cannabinoid ligands. To identify optimal CB1 constructs for these studies (those with the best expression levels, solubility in detergent and function), we first screened various CB1-green fluorescent protein chimeras in a mammalian expression system. Once identified, we then tagged the best candidates with the 1D4 epitope (the C-terminus of rhodopsin) and purified them using a single-step immunoaffinity process. The resulting, highly pure proteins retain their ability to activate G-protein, and are ~85% functional, as assessed by radioligand binding studies. The SDFL studies involve introducing single cysteine residues at key places in the receptor, then labeling them with a small fluorophore, bimane. The spectral properties of the bimane probe are then monitored before and after addition of cannabinoid ligands. Changes in fluorescence of the attached probe indicate regions of the receptor undergoing conformational changes upon ligand binding. Together, these approaches set the stage for a deeper understanding of the structure and function of CB1. Access to pure, functional CB1 makes subsequent structural studies possible (such as crystallography and single-particle EM analysis), and the SDFL studies enable a better structural and mechanistic understanding of this key receptor and the dynamic changes it undergoes during activation and attenuation. © 2017 Elsevier Inc. All rights reserved.

  4. Mapping molecular flexibility of proteins with site-directed spin labeling: a case study of myoglobin.

    Science.gov (United States)

    López, Carlos J; Oga, Shirley; Hubbell, Wayne L

    2012-08-21

    Site-directed spin labeling (SDSL) has potential for mapping protein flexibility under physiological conditions. The purpose of the present study was to explore this potential using 38 singly spin-labeled mutants of myoglobin distributed throughout the sequence. Correlation of the EPR spectra with protein structure provides new evidence that the site-dependent variation in line shape, and hence motion of the spin label, is due largely to differences in mobility of the helical backbone in the ns time range. Fluctuations between conformational substates, typically in the μs-ms time range, are slow on the EPR time scale, and the spectra provide a snapshot of conformational equilibria frozen in time as revealed by multiple components in the spectra. A recent study showed that osmolyte perturbation can positively identify conformational exchange as the origin of multicomponent spectra (López et al. (2009), Protein Sci. 18, 1637). In the present study, this new strategy is employed in combination with line shape analysis and pulsed-EPR interspin distance measurements to investigate the conformation and flexibility of myoglobin in three folded and partially folded states. The regions identified to be in conformational exchange in the three forms agree remarkably well with those assigned by NMR, but the faster time scale of EPR allows characterization of localized states not detected in NMR. Collectively, the results suggest that SDSL-EPR and osmolyte perturbation provide a facile means for mapping the amplitude of fast backbone fluctuations and for detecting sequences in slow conformational exchange in folded and partially folded protein sequences.

  5. Construction, characterization, and mutagenesis of an anti-fluorescein single chain antibody idiotype family.

    Science.gov (United States)

    Denzin, L K; Voss, E W

    1992-05-05

    In addition to crystallographic studies that determined antigen contact residues for monoclonal anti-fluorescein (Fl) antibody 4-4-20 (Ka = 2.5 x 10(10) M-1), primary structure comparisons revealed idiotypically cross-reactive monoclonal antibodies (mAbs) 9-40 (Ka = 4.4 x 10(7) M-1), 12-40 (Ka = 4.0 x 10(8) M-1), and 5-14 (Ka = 2.4 x 10(8) M-1) possessed identical Fl contact residues, with the exception of L34His for L34Arg. Site-specific mutagenesis of single chain antibody (SCA) 4-4-20 in which L34Arg was changed to L34His resulted in approximately 1000- and 3-fold decreases in binding affinity and Qmax (maximum quenching of bound Fl), respectively, which suggested that L34Arg was directly involved in increased binding affinity and fluorescence quenching. Therefore, substitution of Arg for His at residue L34 in mAbs 9-40, 12-40, and 5-14 should result in increased binding affinity and Qmax. To facilitate site-specific mutagenesis studies, single chain derivatives of mAbs 9-40, 12-40, and 5-14 were constructed. Following expression in Escherichia coli, characterization of the SCAs demonstrated that when compared with the respective parental mAb, the SCAs possessed identical binding affinities and similar Qmax and lambda max (absorption profiles of bound Fl) values. These results validated SCA 9-40, 12-40, and 5-14 for use in site-directed mutagenesis studies. Results of mutagenesis studies indicated that substitution of L34Arg into the active sites of 9-40, 12-40, and 5-14 was not enough to produce 4-4-20-like binding characteristics. Therefore, the following single chain mutants were constructed: 9-40L34Arg/L46Val, 12-40L34Arg/L46Val and 5-14L34Arg/L46Val, 9-40L34Arg/L46Val/H101Asp and 4-4-20H101Ala. Results demonstrated that these mutations were not able to render the mutant SCAs with increased binding affinity and fluorescence quenching values. Collectively, these results suggest that the combining sites of mAb 9-40, 12-40, and 5-14 may possess different active

  6. 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

  7. 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.

  8. CRISPR/Cas9-mediated targeted mutagenesis in grape.

    Science.gov (United States)

    Nakajima, Ikuko; Ban, Yusuke; Azuma, Akifumi; Onoue, Noriyuki; Moriguchi, Takaya; Yamamoto, Toshiya; Toki, Seiichi; Endo, Masaki

    2017-01-01

    RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape-an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. 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.

  11. 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.

  12. Mechanisms of retroviral integration and mutagenesis.

    Science.gov (United States)

    Cavazza, Alessia; Moiani, Arianna; Mavilio, Fulvio

    2013-02-01

    Gene transfer vectors derived from oncoretroviruses or lentiviruses are the most robust and reliable tools to stably integrate therapeutic transgenes in human cells for clinical applications. Integration of these vectors in the genome may, however, have undesired effects caused by insertional deregulation of gene expression at the transcriptional or post-transcriptional level. The occurrence of severe adverse events in several clinical trials involving the transplantation of stem cells genetically corrected with retroviral vectors showed that insertional mutagenesis is not just a theoretical event, and that retroviral transgenesis is associated with a finite risk of genotoxicity. In addressing these issues, the gene therapy community offered a spectacular example of how scientific knowledge and technology can be put to work to understand the causes of unpredicted side effects, design new vectors, and develop tools and models to predict their safety and efficacy. As an added benefit, these efforts brought new basic knowledge on virus-host interactions and on the biology and dynamics of human somatic stem cells. This review summarizes the current knowledge on the interactions between retroviruses and the human genome and addresses the impact of target site selection on the safety of retroviral vector-mediated gene therapy.

  13. History of attempts to quantify environmental mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Hollaender, A.

    1981-01-01

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

  14. A targeted mutagenesis system for Actinobacillus pleuropneumoniae.

    Science.gov (United States)

    Mulks, M H; Buysse, J M

    1995-11-07

    We describe methods for the mutagenesis of cloned Actinobacillus pleuropneumoniae (Ap) genes and for the construction of Ap mutants by allelic exchange. We used these methods to construct isogenic mutants of Ap which no longer synthesized a 48-kDa outer membrane protein (AopA). The native aopA locus was replaced with a mutated locus that had been inactivated by insertion of a gene (KmR) encoding kanamycin resistance from Tn903. The inactivated aopA locus was cloned into a conjugative, R6K-derived, lambda pir-dependent suicide vector and introduced into Ap using a filtermating technique. Southern and Western blot analyses indicated that the wild-type locus was replaced by the mutated locus through either single- or double-crossover events, and that AopA was no longer produced by either type of mutant. These methods were used successfully to construct AopA- mutants in Ap serotypes 1 and 5. These methods should be generally useful in constructing mutant loci which can be used to analyze the roles of various Ap genes in the pathogenesis of contagious pleuropneumonia in swine.

  15. Genomic approaches to DNA repair and mutagenesis.

    Science.gov (United States)

    Wyrick, John J; Roberts, Steven A

    2015-12-01

    DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. 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

  17. 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)

  18. 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.

  19. Environmental stress induces trinucleotide repeat mutagenesis in human cells

    Science.gov (United States)

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

    2015-01-01

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

  20. 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.

  1. Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Pletneva, Nadya V.; Pletnev, Vladimir Z., E-mail: vzpletnev@gmail.com; Souslova, Ekaterina; Chudakov, Dmitry M. [Russian Academy of Sciences, Moscow (Russian Federation); Lukyanov, Sergey [Russian Academy of Sciences, Moscow (Russian Federation); Nizhny Novgorod State Medical Academy, Nizhny Novgorod (Russian Federation); Martynov, Vladimir I.; Arhipova, Svetlena; Artemyev, Igor [Russian Academy of Sciences, Moscow (Russian Federation); Wlodawer, Alexander [National Cancer Institute, Frederick, MD 21702 (United States); Dauter, Zbigniew [National Cancer Institute, Argonne, IL 60439 (United States); Pletnev, Sergei [National Cancer Institute, Argonne, IL 60439 (United States); SAIC-Frederick, 9700 South Cass Avenue, Argonne, IL 60439 (United States); Russian Academy of Sciences, Moscow (Russian Federation)

    2013-06-01

    The yellow fluorescent protein phiYFPv with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The yellow fluorescent protein phiYFPv (λ{sub em}{sup max} ≃ 537 nm) with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow–orange range (535–555 nm). Here, the crystal structure of phiYFPv has been determined at 2.05 Å resolution. The ‘yellow’ chromophore formed from the sequence triad Thr65-Tyr66-Gly67 adopts the bicyclic structure typical of fluorophores emitting in the green spectral range. It was demonstrated that perfect antiparallel π-stacking of chromophore Tyr66 and the proximal Tyr203, as well as Val205, facing the chromophore phenolic ring are chiefly responsible for the observed yellow emission of phiYFPv at 537 nm. Structure-based site-directed mutagenesis has been used to identify the key functional residues in the chromophore environment. The obtained results have been utilized to improve the properties of phiYFPv and its homologous monomeric biomarker tagYFP.

  2. 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.

  3. 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.

  4. Targeted Mutagenesis of Guinea Pig Cytomegalovirus Using CRISPR/Cas9-Mediated Gene Editing.

    Science.gov (United States)

    Bierle, Craig J; Anderholm, Kaitlyn M; Wang, Jian Ben; McVoy, Michael A; Schleiss, Mark R

    2016-08-01

    The cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome in Escherichia coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates. The cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infection in vivo Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial

  5. Electrostatic stabilization in a pre-organized polar active site: the catalytic role of Lys-80 in Candida tenuis xylose reductase (AKR2B5) probed by site-directed mutagenesis and functional complementation studies.

    Science.gov (United States)

    Kratzer, Regina; Nidetzky, Bernd

    2005-07-15

    Lys-80 of Candida tenuis xylose reductase (AKR2B5) is conserved throughout the aldo-keto reductase protein superfamily and may prime the nearby Tyr-51 for general acid catalysis to NAD(P)H-dependent carbonyl group reduction. We have examined the catalytic significance of side-chain substitutions in two AKR2B5 mutants, Lys-80-->Ala (K80A) and Asp-46-->Asn Lys-80-->Ala (D46N K80A), using steady-state kinetic analysis and restoration of activity with external amines. Binding of NAD+ (Kd = 24 microM) and NADP+ (Kd = 0.03 microM) was 10- and 40-fold tighter in K80A than the wild-type enzyme, whereas binding of NADH (Kd = 51 microM) and NADPH (Kd = 19 microM) was weakened 2- and 16-fold in this mutant respectively. D46N K80A bound NAD(P)H and NAD(P)+ uniformly approx. 5-fold less tightly than the wild-type enzyme. The second-order rate constant for non-covalent restoration of NADH-dependent reductase activity (kmax/Kamine) by protonated ethylamine was 0.11 M(-1).s(-1) for K80A, whereas no detectable rescue occurred for D46N K80A. After correction for effects of side-chain hydrophobicity, we obtained a linear free energy relationship of log (kmax/Kamine) and amine group pKa (slope = +0.29; r2 = 0.93) at pH 7.0. pH profiles of log (kcat/Km) for carbonyl group reduction by wild-type and D46N K80A revealed identical and kinetically unperturbed pKa values of 8.50 (+/-0.20). Therefore the protonated side chain of Lys-80 is not an essential activator of general acid catalysis by AKR2B5. Stabilized structurally through the salt-link interaction with the negatively charged Asp-46, it is proposed to pull the side chain of Tyr-51 into the catalytic position, leading to a preorganized polar environment of overall neutral charge, in which approximation of uncharged reactive groups is favoured and thus hydride transfer from NAD(P)H is strongly preferred. Lys-80 affects further the directional preference of AKR2B5 for NAD(P)H-dependent reduction by increasing NAD(P)H compared with NAD(P)+-binding selectivity.

  6. 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.

  7. Changing the hydrogen-bonding potential in the DNA binding site of EcoRI by site-directed mutagenesis drastically reduces the enzymatic activity, not, however, the preference of this restriction endonuclease for cleavage within the site-GAATTC-.

    Science.gov (United States)

    Alves, J; Rüter, T; Geiger, R; Fliess, A; Maass, G; Pingoud, A

    1989-03-21

    According to the X-ray structure analysis of an EcoRI-oligodeoxynucleotide complex [McClarin et al. (1986) Science 234, 1526], sequence specificity is mediated by 12 hydrogen bonds, 6 from each of the two identical subunits of the dimeric enzyme to the recognition site -GAATTC-: Arg200 forms two hydrogen bonds with guanine, while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. Changing the hydrogen-bonding potential at the recognition site without perturbing the rest of the interface should lead to the recognition of degenerate sequences [Rosenberg et al. (1987) in Protein Engineering (Oxender, D. L., & Fox, C. F., Eds.) pp 237-250, Liss, New York]. We have shown previously that replacing Glu144 by Gln and Arg145 by Lys affects the activity of the enzyme, not, however, its specificity [Wolfes et al. (1986) Nucleic Acids Res. 14, 9063]. We show now that also the mutation of Arg200 to Lys, the double mutation Glu144Arg145 to GlnLys, and the triple mutation Glu144Arg145Arg200 to GlnLysLys do not lead to a detectable degeneracy of the specificity of cleavage by EcoRI but significantly impair the catalytic activity of this enzyme. A detailed analysis of the steady-state kinetics of cleavage of pUC8 DNA and a tridecadeoxynucleotide substrate demonstrates that the reduction in activity for all DNA binding site mutants investigated so far is mainly due to a decrease in kcat, with the exception of the Arg200 to Lys mutant, which is only impaired in its KM.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. 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

  9. 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.

  10. CRISPR/Cas9 genome editing technique and its application in site-directed genome modification of animals.

    Science.gov (United States)

    Zhou, Jin-wei; Xu, Qi-pin; Yao, Jing; Yu, Shu-min; Cao, Sui-zhong

    2015-10-01

    CRISPR/Cas system, which uses CRISPR RNAs (crRNAs) to guide Cas nuclease to silence invading nucleic acids, is self-defense system against exogenous virus or plasmid in bacteria and archaea. Through molecular modification, the typeⅡCRISPR/Cas system has become a highly efficient site-directed genome editing technique, which is simpler than zinc-finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs) and easier to be designed and applied. In this review, we summarize the evolutionary history of CRISPR/Cas9 system, the working principle and modification process of type Ⅱ CRISPR/Cas and its application in animal genome modification. We also analyze the existing problems and improvement program of the CRISPR/Cas9 system as well as its application prospect combined with successful cases, which may provide innovative perspectives on improving animal traits and establishing animal models of human diseases.

  11. 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.

  12. 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.

  13. Structure-based mutagenesis of Penicillium griseofulvum xylanase using computational design.

    Science.gov (United States)

    André-Leroux, Gwénaëlle; Berrin, Jean-Guy; Georis, Jacques; Arnaut, Filip; Juge, Nathalie

    2008-09-01

    Penicillium griseofulvum xylanase (PgXynA) belongs to family 11 glycoside hydrolase. It exhibits unique amino acid features but its three-dimensional structure is not known. Based upon the X-ray structure of Penicillium funiculosum xylanase (PfXynC), we generated a three-dimensional model of PgXynA by homology modeling. The native structure of PgXynA displayed the overall beta-jelly roll folding common to family 11 xylanases with two large beta-pleated sheets and a single alpha-helix that form a structure resembling a partially closed right hand. Although many features of PgXynA were very similar to previously described enzymes from this family, crucial differences were observed in the loop forming the "thumb" and at the edge of the binding cleft. The robustness of the xylanase was challenged by extensive in silico-based mutagenesis analysis targeting mutations retaining stereochemical and energetical control of the protein folding. On the basis of structural alignments, modeled three-dimensional structure, in silico mutations and docking analysis, we targeted several positions for the replacement of amino acids by site-directed mutagenesis to change substrate and inhibitor specificity, alter pH profile and improve overall catalytic activity. We demonstrated the crucial role played by Ser44(PgXynA) and Ser129(PgXynA), two residues unique to PgXynA, in conferring distinct specificity to P. griseofulvum xylanase. We showed that the pH optimum of PgXynA could be shifted by -1 to +0.5 units by mutating Ser44(PgXynA) to Asp and Asn, respectively. The S44D and S44N mutants showed only slight alteration in K(m) and V(max) whereas a S44A mutant lost both pH-dependence profile and activity. We were able to produce PgXynA S129G mutants with acquired sensitivity to the Xylanase Inhibitor Protein, XIP-I. The replacement of Gln121(PgXynA), located at the start of the thumb, into an Arg residue resulted in an enzyme that possessed a higher catalytic activity. 2008 Wiley

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

    Directory of Open Access Journals (Sweden)

    Ihssen Julian

    2012-09-01

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

  15. 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.

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

    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 ...

  17. 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...

  18. Generation of genetically modified rodents using random ENU mutagenesis

    NARCIS (Netherlands)

    van Boxtel, R.; Cuppen, E.

    2011-01-01

    The generation of genetically modified animals using N-ethyl-N-nitrosourea (ENU) mutagenesis is a fast and highly effective method. The technique is based on treating male animals with the supermutagen ENU, which randomly introduces mutations in the spermatogonial stem cells. By breeding these

  19. 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 ...

  20. 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...

  1. An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.

    Science.gov (United States)

    Paun, Linda; Nitsche, Benjamin; Homan, Tim; Ram, Arthur F; Kempken, Frank

    2016-07-01

    The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background.

  2. 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.

  3. 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

  4. 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.

  5. Mutagenesis of catalytically important residues of cupin type phosphoglucose isomerase from Archaeoglobus fulgidus.

    Science.gov (United States)

    Hansen, Thomas; Schlichting, Bettina; Grötzinger, Joachim; Swan, Michael K; Davies, Christopher; Schönheit, Peter

    2005-12-01

    Recently, cupin type phosphoglucose isomerases have been described as a novel protein family representing a separate lineage in the evolution of phosphoglucose isomerases. The importance of eight active site residues completely conserved within the cPGI family has been assessed by site-directed mutagenesis using the cPGI from Archaeoglobus fulgidus (AfcPGI) as a model. The mutants T63A, G79A, G79L, H80A, H80D, H82A, E93A, E93D, Y95F, Y95K, H136A, and Y160F were constructed, purified, and the impact of the respective mutation on catalysis and/or metal ion binding as well as thermostability was analyzed. The variants G79A, G79L, and Y95F exhibited a lower thermostability. The catalytic efficiency of the enzyme was reduced by more than 100-fold in the G79A, G79L, H80A, H80D, E93D, Y95F variants and more than 15-fold in the T63A, H82A, Y95K, Y160F variants, but remained about the same in the H136A variant at Ni2+ saturating conditions. Further, the Ni2+ content of the mutants H80A, H80D, H82A, E93A, E93D and their apparent Ni2+ binding ability was reduced, resulting in an almost complete loss of activity and thus underlining the crucial role of the metal ion for catalysis. Evidence is presented that H80, H82 and E93 play an additional role in catalysis besides metal ion binding. E93 appears to be the key catalytic residue of AfcPGI, as the E93A mutant did not show any catalytic activity at all.

  6. Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis

    Science.gov (United States)

    Bulina, Maria E; Chudakov, Dmitry M; Mudrik, Nikolay N; Lukyanov, Konstantin A

    2002-01-01

    Background Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date. Results Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein. Conclusions We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed. PMID:11972899

  7. Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis

    Directory of Open Access Journals (Sweden)

    Mudrik Nikolay N

    2002-04-01

    Full Text Available Abstract Background Within the family of green fluorescent protein (GFP homologs, one can mark two main groups, specifically, fluorescent proteins (FPs and non-fluorescent or chromoproteins (CPs. Structural background of differences between FPs and CPs are poorly understood to date. Results Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595 from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield ( Conclusions We located a novel point in asCP sequence (position 165 mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed.

  8. Discovery of novel STAT3 small molecule inhibitors via in silico site-directed fragment-based drug design.

    Science.gov (United States)

    Yu, Wenying; Xiao, Hui; Lin, Jiayuh; Li, Chenglong

    2013-06-13

    Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been validated as an attractive therapeutic target for cancer therapy. To stop both STAT3 activation and dimerization, a viable strategy is to design inhibitors blocking its SH2 domain phosphotyrosine binding site that is responsible for both actions. A new fragment-based drug design (FBDD) strategy, in silico site-directed FBDD, was applied in this study. A designed novel compound, 5,8-dioxo-6-(pyridin-3-ylamino)-5,8-dihydronaphthalene-1-sulfonamide (LY5), was confirmed to bind to STAT3 SH2 by fluorescence polarization assay. In addition, four out of the five chosen compounds have IC50 values lower than 5 μM for the U2OS cancer cells. 8 (LY5) has an IC50 range in 0.5-1.4 μM in various cancer cell lines. 8 also suppresses tumor growth in an in vivo mouse model. This study has demonstrated the utility of this approach and could be used to other drug targets in general.

  9. SimLabel: a graphical user interface to simulate continuous wave EPR spectra from site-directed spin labeling experiments.

    Science.gov (United States)

    Etienne, E; Le Breton, N; Martinho, M; Mileo, E; Belle, V

    2017-08-01

    Site-directed spin labeling (SDSL) combined with continuous wave electron paramagnetic resonance (cw EPR) spectroscopy is a powerful technique to reveal, at the residue level, structural transitions in proteins. SDSL-EPR is based on the selective grafting of a paramagnetic label on the protein under study, followed by cw EPR analysis. To extract valuable quantitative information from SDSL-EPR spectra and thus give reliable interpretation on biological system dynamics, numerical simulations of the spectra are required. Such spectral simulations can be carried out by coding in MATLAB using functions from the EasySpin toolbox. For non-expert users of MATLAB, this could be a complex task or even impede the use of such simulation tool. We developed a graphical user interface called SimLabel dedicated to run cw EPR spectra simulations particularly coming from SDSL-EPR experiments. Simlabel provides an intuitive way to visualize, simulate, and fit such cw EPR spectra. An example of SDSL-EPR spectra simulation concerning the study of an intrinsically disordered region undergoing a local induced folding is described and discussed. We believe that this new tool will help the users to rapidly obtain reliable simulated spectra and hence facilitate the interpretation of their results. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  10. 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.

  11. 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.

  12. 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.

  13. UV-Induced DNA Damage and Mutagenesis in Chromatin.

    Science.gov (United States)

    Mao, Peng; Wyrick, John J; Roberts, Steven A; Smerdon, Michael J

    2017-01-01

    UV radiation induces photolesions that distort the DNA double helix and, if not repaired, can cause severe biological consequences, including mutagenesis or cell death. In eukaryotes, both the formation and repair of UV damage occur in the context of chromatin, in which genomic DNA is packaged with histones into nucleosomes and higher order chromatin structures. Here, we review how chromatin impacts the formation of UV photoproducts in eukaryotic cells. We describe the initial discovery that nucleosomes and other DNA binding proteins induce characteristic "photofootprints" during the formation of UV photoproducts. We also describe recent progress in genomewide methods for mapping UV damage, which echoes early biochemical studies, and highlights the role of nucleosomes and transcription factors in UV damage formation and repair at unprecedented resolution. Finally, we discuss our current understanding of how the distribution and repair of UV-induced DNA damage influence mutagenesis in human skin cancers. © 2016 The American Society of Photobiology.

  14. Oligonucleotide?directed mutagenesis for precision gene editing

    OpenAIRE

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

    2015-01-01

    Summary 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 a...

  15. 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.

  16. GATMD: γ-Aminobutyric Acid Transporter Mutagenesis Database

    Science.gov (United States)

    Anderson, Cynthia M.; Kidd, Patrick D.; Eskandari, Sepehr

    2010-01-01

    Since the cloning of the first γ-aminobutyric acid (GABA) transporter (GAT1; SLC6A1) from rat brain in 1990, more than 50 published studies have provided structure–function information on investigator-designed rat and mouse GAT1 mutants. To date, more than 200 of 599 GAT1 residues have been subjected to mutagenesis experiments by substitution with different amino acids, and the resulting transporter functional properties have significantly advanced our understanding of the mechanism of Na+- and Cl–-coupled GABA transport by this important member of the neurotransmitter:sodium symporter family. Moreover, many studies have addressed the functional consequences of amino acid deletion or insertion at various positions along the primary sequence. The enormity of this growing body of structure–function information has prompted us to develop GABA Transporter Mutagenesis Database (GATMD), a web-accessible, relational database of manually annotated biochemical, functional and pharmacological data reported on GAT1—the most intensely studied GABA transporter isoform. As of the last update of GATMD, 52 GAT1 mutagenesis papers have yielded 3360 experimental records, which collectively contain a total of ∼100 000 annotated parameters. Database URL: http://physiology.sci.csupomona.edu/GATMD/ PMID:21131297

  17. 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

  18. Coherent wavepackets in the Fenna-Matthews-Olson complex are robust to excitonic-structure perturbations caused by mutagenesis

    Science.gov (United States)

    Maiuri, Margherita; Ostroumov, Evgeny E.; Saer, Rafael G.; Blankenship, Robert E.; Scholes, Gregory D.

    2018-02-01

    Femtosecond pulsed excitation of light-harvesting complexes creates oscillatory features in their response. This phenomenon has inspired a large body of work aimed at uncovering the origin of the coherent beatings and possible implications for function. Here we exploit site-directed mutagenesis to change the excitonic level structure in Fenna-Matthews-Olson (FMO) complexes and compare the coherences using broadband pump-probe spectroscopy. Our experiments detect two oscillation frequencies with dephasing on a picosecond timescale—both at 77 K and at room temperature. By studying these coherences with selective excitation pump-probe experiments, where pump excitation is in resonance only with the lowest excitonic state, we show that the key contributions to these oscillations stem from ground-state vibrational wavepackets. These experiments explicitly show that the coherences—although in the ground electronic state—can be probed at the absorption resonances of other bacteriochlorophyll molecules because of delocalization of the electronic excitation over several chromophores.

  19. Coherent wavepackets in the Fenna-Matthews-Olson complex are robust to excitonic-structure perturbations caused by mutagenesis.

    Science.gov (United States)

    Maiuri, Margherita; Ostroumov, Evgeny E; Saer, Rafael G; Blankenship, Robert E; Scholes, Gregory D

    2018-02-01

    Femtosecond pulsed excitation of light-harvesting complexes creates oscillatory features in their response. This phenomenon has inspired a large body of work aimed at uncovering the origin of the coherent beatings and possible implications for function. Here we exploit site-directed mutagenesis to change the excitonic level structure in Fenna-Matthews-Olson (FMO) complexes and compare the coherences using broadband pump-probe spectroscopy. Our experiments detect two oscillation frequencies with dephasing on a picosecond timescale-both at 77 K and at room temperature. By studying these coherences with selective excitation pump-probe experiments, where pump excitation is in resonance only with the lowest excitonic state, we show that the key contributions to these oscillations stem from ground-state vibrational wavepackets. These experiments explicitly show that the coherences-although in the ground electronic state-can be probed at the absorption resonances of other bacteriochlorophyll molecules because of delocalization of the electronic excitation over several chromophores.

  20. 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.

  1. p21-ras effector domain mutants constructed by "cassette" mutagenesis

    DEFF Research Database (Denmark)

    Stone, J C; Vass, W C; Willumsen, B M

    1988-01-01

    A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis...... technique that involved replacing this portion of the v-rasH effector domain with a linker carrying two BspMI sites in opposite orientations. Since BspMI cleaves outside its recognition sequence, BspMI digestion of the plasmid completely removed the linker, creating a double-stranded gap whose missing ras...

  2. A protocol for chemical mutagenesis in Strongyloides ratti.

    Science.gov (United States)

    Guo, Li; Chang, Zisong; Dieterich, Christoph; Streit, Adrian

    2015-11-01

    Genetic analysis using experimentally induced mutations has been a most valuable tool in the analysis of various organisms. However, genetic analysis of endoparasitic organisms tends to be difficult because of the limited accessibility of the sexually reproducing adults, which are normally located within the host. Nematodes of the genera Strogyloides and Parastrongyloides represent an exception to this because they can form facultative free-living sexually reproducing generations in between parasitic generations. Here we present a protocol for the chemical mutagenesis of Strongyloides ratti. Further we evaluate the feasibility of identifying the induced mutations by whole genome re-sequencing. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Environmental stress and mutagenesis in enteric and non-enteric bacteria

    OpenAIRE

    Babudri, Nora; Lancioni, Hovirag; Achilli, Alessandro

    2012-01-01

    Mutations are fundamental for evolution. For many years it has been thought that mutagenesis occurs only in dividing cells. Now it is clear that mutations arise in non-dividing or slowly dividing microorganisms. Natural populations spend most of the time in stressful environments where their growth rate is highly reduced. Thus, the existence of a mutagenesis process, independent of multiplication (stress-induced mutagenesis, SIM), might have a profound evolutionary role. In the presented pape...

  4. Increased sensitivity to CD4 binding site-directed neutralization following in vitro propagation on primary lymphocytes of a neutralization-resistant human immunodeficiency virus IIIB strain isolated from an accidentally infected laboratory worker.

    Science.gov (United States)

    Beaumont, Tim; Quakkelaar, Esther; van Nuenen, Ad; Pantophlet, Ralph; Schuitemaker, Hanneke

    2004-06-01

    We previously described the adaptation of the neutralization-sensitive human immunodeficiency virus type 1 (HIV-1) strain IIIB to a neutralization-resistant phenotype in an accidentally infected laboratory worker. During long-term propagation of this resistant isolate, designated FF3346, on primary peripheral blood leukocytes in vitro, an HIV-1 variant appeared that had regained sensitivity to neutralization by soluble CD4 (sCD4) and the broadly neutralizing monoclonal antibody b12. When an early passage of FF3346 was subjected to limiting-dilution culture in peripheral blood mononuclear cells, eight virus variants with various degrees of neutralization resistance were isolated. Two of them, the sCD4 neutralization-resistant variant LW_H8(res) and the sCD4 neutralization-sensitive variant LW_G9(sens), were selected for further study. Interestingly, these two viruses were equally resistant to neutralization by agents that recognize domains other than the CD4 binding site. Site-directed mutagenesis revealed that the increased neutralization sensitivity of variant LW_G9(sens) resulted from only two changes, an Asn-to-Ser substitution at position 164 in the V2 loop and an Ala-to-Glu substitution at position 370 in the C3 domain of gp120. In agreement with this notion, the affinity of b12 for monomeric gp120 containing the N164S and A370E substitutions in the background of the molecular clone LW_H8(res) was higher than its affinity for the parental gp120. Surprisingly, no correlation was observed between CD4 binding affinity for monomeric gp120 and the level of neutralization resistance, suggesting that differences in sCD4 neutralization sensitivity between these viruses are only manifested in the context of the tertiary or quaternary structure of gp120 on the viral surface. The results obtained here indicate that the neutralization-sensitive strain IIIB can become neutralization resistant in vivo under selective pressure by neutralizing antibodies but that this

  5. New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis.

    Science.gov (United States)

    Kuroyanagi, Miwa; Katayama, Takashi; Imai, Tadashi; Yamamoto, Yoshihisa; Chisada, Shin-ichi; Yoshiura, Yasutoshi; Ushijima, Tomokazu; Matsushita, Tomonao; Fujita, Masashi; Nozawa, Aoi; Suzuki, Yuzuru; Kikuchi, Kiyoshi; Okamoto, Hiroyuki

    2013-11-13

    In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported. In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING

  6. Cell-mediated mutagenesis and cell transformation by chemical carcinogens

    Energy Technology Data Exchange (ETDEWEB)

    Huberman, E.; Langenbach, R.

    1977-01-01

    Results are reported from studies that showed that mutagenesis of mammalian cells can be achieved by carcinogenic polycyclic hydrocarbons, nitrosamines, and aflatoxins when tested in the presence of fibroblasts and hepatocytes which are able to metabolize these carcinogens. Further, we have found that there is a relationship between the degree of mutant induction and the degree of carcinogenicity of the different chemicals tested. By simultaneously measuring the frequency of cell transformation and the frequency of mutation at one locus (ouabain resistance) in the same cell system, it was possible to estimate the genetic target site for cell transformation. The results indicated that the target site for transformation is approximately 20 times larger than that determined for ouabain resistance. The results suggest that cell transformation may be due to a mutational event and the mutation can occur in one out of a small number of the same or different genes, and that the cell-mediated mutagenesis approach may be a valuable means of detecting tissue-specific carcinogens.

  7. Mariner-based transposon mutagenesis for Bacteroides species.

    Science.gov (United States)

    Ichimura, Minoru; Uchida, Keiko; Nakayama-Imaohji, Haruyuki; Hirakawa, Hideki; Tada, Tomoyo; Morita, Hidetoshi; Yasutomo, Koji; Okazaki, Katsuichiro; Kuwahara, Tomomi

    2014-06-01

    Bacteroides is one of the most predominant groups of human gut microbiota. Recent metagenomic analyses and studies on gnotobiotic mice demonstrated the tight association of Bacteroides with epithelial function, the gut immune system and systemic metabolism in the host. The mariner family transposon shows relatively low target site specificity and has hosts ranging from prokaryotes to eukaryotes. Thereby, random mutagenesis using the mariner family transposon is expected to identify key molecules for human-Bacteroides symbiosis. In this study, we constructed the plasmid pMI07 to deliver the gene cassette (ermF/ITR), which harbors the erythromycin resistant marker (ermF) and the inverted repeat sequences (ITRs) recognized by Himar1 transposase, to Bacteroides via electrotransformation. pMI07 successfully delivered ermF/ITR to the Bacteroides genomes and generated thousands of insertion mutants/μg of pMI07 in B. thetaiotaomicron, B. fragilis, B. ovatus, and also, although to a lesser extent, B. vulgatus. Analyses of the ermF/ITR insertion sites in B. thetaiotaomicron and B. vulgatus revealed that the cassette targeted the dinucleotide TA and integrated into the genomes in an unbiased manner. The data reported here will provide useful information for transposon mutagenesis in Bacteroides species, which will enable identification of the genes responsible for their unique phenotypes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Lethal Mutagenesis of Hepatitis C Virus Induced by Favipiravir.

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    Ana I de Ávila

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

  9. 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

  10. Site-Directed Mutation, Cloning and Expression of Streptokinase for Producing a New Suitable Molecule for PEGylation

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    Somayeh Bagheri

    2016-06-01

    Full Text Available Abstract Background: Streptokinase is one of the most common and cost effective fibrinolytic drugs for treatment of heart attacks and vein thrombosis. Unlike many advantages over other thrombolytic drugs, administration of streptokinase can produce some complications such as immunologic reactions, hemorrhage and incomplete treatment due to relative short half life. Pegylation is one of the most common methods for improving of these shortcomings. Materials and Methods: In this study, designing a proper candidate for specific pegylation with cysteine was done by means of SPDBviewer software. After a meaning ful mutation by SOEing PCR method, mutated (sk45cys and intact SK (ski genes were cloned in pET26-b vector and the structures were transformed in E.coli. Clones, Afrer growing, were expressed by IpTG and exptression of proteins was confirmed by SDS-PAGE and western blotting. The proteins were purified by affinity chromatography with NiNTA columns and amidolytic activity of purified proteins was assayed using chromogenic method and different concentrations of S2251 substrate. Results: Results of activity assays showed that amidolytic activity of SK45cys had about 10% increase in comparison to Ski, after 30 minutes of complex formation with plasminogen. Conclusion: Generally, it was concluded that, considering cys45 as a superficial aminoacid and also relative increase of activity, SK45cys can be considered a suitable protein for specific pegylation.

  11. Quantitative studies of the mutagenesis of Toxoplasma gondii

    Energy Technology Data Exchange (ETDEWEB)

    Pfefferkorn, E.R.; Pfefferkorn, L.C.

    1979-06-01

    The induction of mutants resistant to 5-fluorodeoxyuridine (FUDR) was used to measure the efficiency of various physical and chemical mutagens on extracellular and intracellular Toxoplasma gondii. The frequency of resistant mutant was measured by plaque assay in human fibroblast cultures in the presence and absence of FUDR. When considered as a function of lethality, the most efficient mutagenesis was obtained with nitrosoguanidine treatment of extracellular parasites and with ethylmethane sulfonate treatment of actively growing intracellular parasites. Each of these treatments increased the frequency of FUDR-resistant mutants from less than one to more than 200 per million parasites. Ultraviolet irradiation, X-rays, and the alkylating mustard ICR-191 also induced FUDR-resistant mutants in a dose-dependent fashion.

  12. Role of Nicotinamide in DNA Damage, Mutagenesis, and DNA Repair

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    Devita Surjana

    2010-01-01

    Full Text Available Nicotinamide is a water-soluble amide form of niacin (nicotinic acid or vitamin B3. Both niacin and nicotinamide are widely available in plant and animal foods, and niacin can also be endogenously synthesized in the liver from dietary tryptophan. Nicotinamide is also commercially available in vitamin supplements and in a range of cosmetic, hair, and skin preparations. Nicotinamide is the primary precursor of nicotinamide adenine dinucleotide (NAD+, an essential coenzyme in ATP production and the sole substrate of the nuclear enzyme poly-ADP-ribose polymerase-1 (PARP-1. Numerous in vitro and in vivo studies have clearly shown that PARP-1 and NAD+ status influence cellular responses to genotoxicity which can lead to mutagenesis and cancer formation. This paper will examine the role of nicotinamide in the protection from carcinogenesis, DNA repair, and maintenance of genomic stability.

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

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    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.

  14. Photo-Oxidative Stress-Driven Mutagenesis and Adaptive Evolution on the Marine Diatom Phaeodactylum tricornutum for Enhanced Carotenoid Accumulation

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    Zhiqian Yi

    2015-09-01

    Full Text Available Marine diatoms have recently gained much attention as they are expected to be a promising resource for sustainable production of bioactive compounds such as carotenoids and biofuels as a future clean energy solution. To develop photosynthetic cell factories, it is important to improve diatoms for value-added products. In this study, we utilized UVC radiation to induce mutations in the marine diatom Phaeodactylum tricornutum and screened strains with enhanced accumulation of neutral lipids and carotenoids. Adaptive laboratory evolution (ALE was also used in parallel to develop altered phenotypic and biological functions in P. tricornutum and it was reported for the first time that ALE was successfully applied on diatoms for the enhancement of growth performance and productivity of value-added carotenoids to date. Liquid chromatography-mass spectrometry (LC-MS was utilized to study the composition of major pigments in the wild type P. tricornutum, UV mutants and ALE strains. UVC radiated strains exhibited higher accumulation of fucoxanthin as well as neutral lipids compared to their wild type counterpart. In addition to UV mutagenesis, P. tricornutum strains developed by ALE also yielded enhanced biomass production and fucoxanthin accumulation under combined red and blue light. In short, both UV mutagenesis and ALE appeared as an effective approach to developing desired phenotypes in the marine diatoms via electromagnetic radiation-induced oxidative stress.

  15. Mechanism of porcine liver xanthine oxidoreductase mediated N-oxide reduction of cyadox as revealed by docking and mutagenesis studies.

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    Chigang Chen

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

  16. 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.

  17. The Yeast Environmental Stress Response Regulates Mutagenesis Induced by Proteotoxic Stress

    Science.gov (United States)

    Shor, Erika; Fox, Catherine A.; Broach, James R.

    2013-01-01

    Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR) in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors. PMID:23935537

  18. The yeast environmental stress response regulates mutagenesis induced by proteotoxic stress.

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    Erika Shor

    Full Text Available Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors.

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

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    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.

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

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    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

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

    Science.gov (United States)

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

    2004-12-01

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

  2. Efficiency and Inheritance of Targeted Mutagenesis in Maize Using CRISPR-Cas9.

    Science.gov (United States)

    Zhu, Jinjie; Song, Ning; Sun, Silong; Yang, Weilong; Zhao, Haiming; Song, Weibin; Lai, Jinsheng

    2016-01-20

    CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently, it has been developed as a powerful targeted genome editing tool for a wide variety of species. However, its application in maize has only been tested with transiently expressed somatic cells or with a limited number of stable transgenic T0 plants. The exact efficiency and specificity of the CRISPR/Cas system in the highly complex maize genome has not been documented yet. Here we report an extensive study of the well-studied type II CRISPR-Cas9 system for targeted genome editing in maize, with the codon-optimized Cas9 protein and the short non-coding guide RNA generated through a functional maize U6 snRNA promoter. Targeted gene mutagenesis was detected for 90 loci by maize protoplast assay, with an average cleavage efficiency of 10.67%. Stable knockout transformants for maize phytoene synthase gene (PSY1) were obtained. Mutations occurred in germ cells can be stably inherited to the next generation. Moreover, no off-target effect was detected at the computationally predicted putative off-target loci. No significant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was detected. Our results confirmed that the CRISPR-Cas9 could be successfully applied as a robust targeted genome editing system in maize. Copyright © 2015 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

  3. Mutagenesis and Genome Engineering of Epstein-Barr Virus in Cultured Human Cells by CRISPR/Cas9.

    Science.gov (United States)

    Yuen, Kit-San; Chan, Chi-Ping; Kok, Kin-Hang; Jin, Dong-Yan

    2017-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 nuclease (Cas9) system is a powerful genome-editing tool for both chromosomal and extrachromosomal DNA. DNA viruses such as Epstein-Barr virus (EBV), which undergoes episomal replication in human cells, can be effectively edited by CRISPR/Cas9. We have demonstrated targeted editing of the EBV genome by CRISPR/Cas9 in several lines of EBV-infected cells. CRISPR/Cas9-based mutagenesis and genome engineering of EBV provides a new method for genetic analysis, which has some advantages over bacterial artificial chromosome-based recombineering. This approach might also prove useful in the cure of EBV infection. In this chapter, we use the knockout of the BART promoter as an example to detail the experimental procedures for construction of recombinant EBV in human cells.

  4. Dimerization and enzymatic activity of fungal 17β-hydroxysteroid dehydrogenase from the short-chain dehydrogenase/reductase superfamily

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    Kristan Katja

    2005-12-01

    Full Text Available Abstract Background 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl is a member of the short-chain dehydrogenase/reductase (SDR superfamily. SDR proteins usually function as dimers or tetramers and 17β-HSDcl is also a homodimer under native conditions. Results We have investigated here which secondary structure elements are involved in the dimerization of 17β-HSDcl and examined the importance of dimerization for the enzyme activity. Sequence similarity with trihydroxynaphthalene reductase from Magnaporthe grisea indicated that Arg129 and His111 from the αE-helices interact with the Asp121, Glu117 and Asp187 residues from the αE and αF-helices of the neighbouring subunit. The Arg129Asp and His111Leu mutations both rendered 17β-HSDcl monomeric, while the mutant 17β-HSDcl-His111Ala was dimeric. Circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. The three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of NADP+, but both monomers showed no enzymatic activity. Conclusion We have shown by site-directed mutagenesis and structure/function analysis that 17β-HSDcl dimerization involves the αE and αF helices of both subunits. Neighbouring subunits are connected through hydrophobic interactions, H-bonds and salt bridges involving amino acid residues His111 and Arg129. Since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. A detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor.

  5. 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.

  6. Combinatorial Mutagenesis and Selection to Understand and Improve Yeast Promoters

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    Laila Berg

    2013-01-01

    Full Text Available Microbial promoters are important targets both for understanding the global gene expression and developing genetic tools for heterologous expression of proteins and complex biosynthetic pathways. Previously, we have developed and used combinatorial mutagenesis methods to analyse and improve bacterial expression systems. Here, we present for the first time an analogous strategy for yeast. Our model promoter is the strong and inducible promoter in methylotrophic Pichia pastoris. The Zeocin resistance gene was applied as a valuable reporter for mutant promoter activity, and we used an episomal plasmid vector to ensure a constant reporter gene dosage in the yeast host cells. This novel design enabled direct selection for colonies of recombinant cells with altered Zeocin tolerance levels originating solely from randomly introduced point mutations in the promoter DNA sequence. We demonstrate that this approach can be used to select for promoter variants with abolished glucose repression in large mutant libraries. We also selected promoter variants with elevated expression level under induced conditions. The properties of the selected promoter variants were confirmed by expressing luciferase as an alternative reporter gene. The tools developed here should be useful for effective screening, characterization, and improvement of any yeast promoters.

  7. 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. PMID:24363725

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. Genome-wide transposon mutagenesis in Saccharomyces cerevisiae and Candida albicans.

    Science.gov (United States)

    Xu, Tao; Bharucha, Nikë; Kumar, Anuj

    2011-01-01

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

  14. 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.

  15. The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences.

    Science.gov (United States)

    Donnelly, M L; Hughes, L E; Luke, G; Mendoza, H; ten Dam, E; Gani, D; Ryan, M D

    2001-05-01

    The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins 'cleaving' at their own C termini. We have analysed this activity using artificial reporter polyprotein systems comprising green fluorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A to beta-glucuronidase (GUS) -- forming a single, long, open reading frame. Analysis of the distribution of radiolabel showed a high proportion of the in vitro translation products (approximately 90%) were in the form of the 'cleavage' products GUS and [GFP2A]. Alternative models have been proposed to account for the 'cleavage' activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect. To investigate the mechanism of this cleavage event constructs encoding site-directed mutant and naturally occurring '2A-like' sequences were used to program in vitro translation systems and the gel profiles analysed. Analysis of site-directed mutant 2A sequences showed that 'cleavage' occurred in constructs in which all the candidate nucleophilic residues were substituted -- with the exception of aspartate-12. This residue is not, however, conserved amongst all functional '2A-like' sequences. '2A-like' sequences were identified within insect virus polyproteins, the NS34 protein of type C rotaviruses, repeated sequences in Trypanosoma spp. and a eubacterial alpha-glucosiduronasesequence(Thermatoga maritima aguA). All of the 2A-like sequences analysed were active (to various extents), other than the eubacterial alpha-glucosiduronase 2A-like sequence. This method of control of protein biogenesis may well not, therefore, be confined to members of the PICORNAVIRIDAE: Taken together, these data provide additional evidence that neither FMDV 2A nor '2A-like' sequences are autoproteolytic elements.

  16. Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development

    Science.gov (United States)

    Liu, Chia-Lin; Hung, Hui-Chen; Lo, Shou-Chen; Chiang, Ching-Hui; Chen, I.-Jung; Hsu, John T.-A.; Hou, Ming-Hon

    2016-02-01

    Nucleoprotein (NP) is the most abundant type of RNA-binding viral protein in influenza A virus-infected cells and is necessary for viral RNA transcription and replication. Recent studies demonstrated that influenza NP is a valid target for antiviral drug development. The surface of the groove, covered with numerous conserved residues between the head and body domains of influenza A NP, plays a crucial role in RNA binding. To explore the mechanism by which NP binds RNA, we performed a series of site-directed mutagenesis in the RNA-binding groove, followed by surface plasmon resonance (SPR), to characterize the interactions between RNA and NP. Furthermore, a role of Y148 in NP stability and NP-RNA binding was evaluated. The aromatic residue of Y148 was found to stack with a nucleotide base. By interrupting the stacking interaction between Y148 and an RNA base, we identified an influenza virus NP inhibitor, (E, E)-1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione; this inhibitor reduced the NP’s RNA-binding affinity and hindered viral replication. Our findings will be useful for the development of new drugs that disrupt the interaction between RNA and viral NP in the influenza virus.

  17. In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyo Joon; Nishikawa, Satoshi; Tokutomi, Yuiko; Uesugi, Seiichi (Osaka Univ. (Japan)); Takenaka, Hitoshi; Hamada, Minoru (Miyazaki Medical College (Japan)); Kuby, S.A. (Univ. of Utah, Salt Lake City (USA))

    1990-02-06

    Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP{sup 2{minus}} and for AMP{sup 2{minus}} in human cytosolic adenylate kinase, the authors have investigated the enzymic effects of replacement of the arginine residues, which had been assumed by Pai et al. to interact with the phosphoryl groups of AMP{sup 2{minus}} and MgATP{sup 2{minus}}. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the K{sub m,app} values for AMP{sup 2{minus}} of the mutant enzymes, the relatively small increases in the K{sub m,app} values for MgATP{sup 2{minus}}, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. have been reversed and that their ATP-binding site may be assigned as the AMP site.

  18. Mutagenesis of the yellow fever virus NS2B/3 cleavage site: determinants of cleavage site specificity and effects on polyprotein processing and viral replication.

    Science.gov (United States)

    Chambers, T J; Nestorowicz, A; Rice, C M

    1995-03-01

    The determinants of cleavage site specificity of the yellow fever virus (YF) NS3 proteinase for its 2B/3 cleavage site have been studied by using site-directed mutagenesis. Mutations at residues within the GARR decreases S sequence were tested for effects on cis cleavage of an NS2B-3(181) polyprotein during cell-free translation. At the P1 position, only the conservative substitution R-->K exhibited significant levels of cleavage. Conservative and nonconservative substitutions were tolerated at the P1' and P2 positions, resulting in intermediate levels of cleavage. Substitutions at the P3 and P4 positions had no effects on cleavage efficiency in the cell-free assay. Processing at other dibasic sites was studied by using transient expression of a sig2A-5(356) polyprotein. Cleavage at the 2B/3 site was not required for processing at downstream sites. However, increased accumulation of high-molecular-weight viral polyproteins was generally observed for mutations which reduced cleavage efficiency at the 2B/3 site. Several mutations were also tested for their effects on viral replication. Virus was not recovered from substitutions which blocked or substantially reduced cleavage in the cell-free assay, suggesting that efficient cleavage at the 2B/3 site is required for flavivirus replication.

  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. Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis, but enhances DNA polymerase ζ - dependent spontaneous mutagenesis.

    Science.gov (United States)

    Stepchenkova, E I; Tarakhovskaya, E R; Siebler, H M; Pavlov, Y I

    2017-01-01

    Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ. Copyright © 2016

  1. Site-specific genomic (SSG and random domain-localized (RDL mutagenesis in yeast

    Directory of Open Access Journals (Sweden)

    Honigberg Saul M

    2004-04-01

    Full Text Available Abstract Background A valuable weapon in the arsenal available to yeast geneticists is the ability to introduce specific mutations into yeast genome. In particular, methods have been developed to introduce deletions into the yeast genome using PCR fragments. These methods are highly efficient because they do not require cloning in plasmids. Results We have modified the existing method for introducing deletions in the yeast (S. cerevisiae genome using PCR fragments in order to target point mutations to this genome. We describe two PCR-based methods for directing point mutations into the yeast genome such that the final product contains no other disruptions. In the first method, site-specific genomic (SSG mutagenesis, a specific point mutation is targeted into the genome. In the second method, random domain-localized (RDL mutagenesis, a mutation is introduced at random within a specific domain of a gene. Both methods require two sequential transformations, the first transformation integrates the URA3 marker into the targeted locus, and the second transformation replaces URA3 with a PCR fragment containing one or a few mutations. This PCR fragment is synthesized using a primer containing a mutation (SSG mutagenesis or is synthesized by error-prone PCR (RDL mutagenesis. In SSG mutagenesis, mutations that are proximal to the URA3 site are incorporated at higher frequencies than distal mutations, however mutations can be introduced efficiently at distances of at least 500 bp from the URA3 insertion. In RDL mutagenesis, to ensure that incorporation of mutations occurs at approximately equal frequencies throughout the targeted region, this region is deleted at the same time URA3 is integrated. Conclusion SSG and RDL mutagenesis allow point mutations to be easily and efficiently incorporated into the yeast genome without disrupting the native locus.

  2. Short tunnels.

    NARCIS (Netherlands)

    Schreuder, D.A.

    1965-01-01

    Before dealing with the question of lighting short tunnels, it is necessary define what is meant by a tunnel and when it should be called 'short'. Confined to motorized road traffic the following is the most apt definition of a tunnel: every form of roofing-over a road section, irrespective of it

  3. 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.

  4. 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.

  5. MDC-Analyzer: a novel degenerate primer design tool for the construction of intelligent mutagenesis libraries with contiguous sites

    National Research Council Canada - National Science Library

    Tang, Lixia; Wang, Xiong; Ru, Beibei; Sun, Hengfei; Huang, Jian; Gao, Hui

    2014-01-01

    ...) for the automated design of intelligent mutagenesis libraries that can completely cover user-defined randomized sequences, especially when multiple contiguous and/or adjacent sites are targeted...

  6. Abundant off-target edits from site-directed RNA editing can be reduced by nuclear localization of the editing enzyme.

    Science.gov (United States)

    Vallecillo-Viejo, Isabel C; Liscovitch-Brauer, Noa; Montiel-Gonzalez, Maria Fernanda; Eisenberg, Eli; Rosenthal, Joshua J C

    2017-11-03

    Site-directed RNA editing (SDRE) is a general strategy for making targeted base changes in RNA molecules. Although the approach is relatively new, several groups, including our own, have been working on its development. The basic strategy has been to couple the catalytic domain of an adenosine (A) to inosine (I) RNA editing enzyme to a guide RNA that is used for targeting. Although highly efficient on-target editing has been reported, off-target events have not been rigorously quantified. In this report we target premature termination codons (PTCs) in messages encoding both a fluorescent reporter protein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein transiently transfected into human epithelial cells. We demonstrate that while on-target editing is efficient, off-target editing is extensive, both within the targeted message and across the entire transcriptome of the transfected cells. By redirecting the editing enzymes from the cytoplasm to the nucleus, off-target editing is reduced without compromising the on-target editing efficiency. The addition of the E488Q mutation to the editing enzymes, a common strategy for increasing on-target editing efficiency, causes a tremendous increase in off-target editing. These results underscore the need to reduce promiscuity in current approaches to SDRE.

  7. A new lead for nonpeptidic active-site-directed inhibitors of the severe acute respiratory syndrome coronavirus main protease discovered by a combination of screening and docking methods.

    Science.gov (United States)

    Kaeppler, Ulrich; Stiefl, Nikolaus; Schiller, Markus; Vicik, Radim; Breuning, Alexander; Schmitz, Werner; Rupprecht, Daniel; Schmuck, Carsten; Baumann, Knut; Ziebuhr, John; Schirmeister, Tanja

    2005-11-03

    The coronavirus main protease, M(pro), is considered to be a major target for drugs suitable for combating coronavirus infections including severe acute respiratory syndrome (SARS). An HPLC-based screening of electrophilic compounds that was performed to identify potential M(pro) inhibitors revealed etacrynic acid tert-butylamide (6a) as an effective nonpeptidic inhibitor. Docking studies suggested a binding mode in which the phenyl ring acts as a spacer bridging the inhibitor's activated double bond and its hydrophobic tert-butyl moiety. The latter is supposed to fit into the S4 pocket of the target protease. Furthermore, these studies revealed etacrynic acid amide (6b) as a promising lead for nonpeptidic active-site-directed M(pro) inhibitors. In a fluorimetric enzyme assay using a novel fluorescence resonance energy transfer (FRET) pair labeled substrate, compound 6b showed a K(i) value of 35.3 muM. Since the novel lead compound does not target the S1', S1, and S2 subsites of the enzyme's substrate-binding pockets, there is room for improvement that underlines the lead character of compound 6b.

  8. Identifying the antiasthmatic target of doxofylline using immobilized β2 -adrenoceptor based high-performance affinity chromatography and site-directed molecular docking.

    Science.gov (United States)

    Zhang, Yajun; Zeng, Kaizhu; Wang, Jing; Gao, Haiyang; Nan, Yefei; Zheng, Xiaohui

    2016-10-01

    As a xanthine derivative, doxofylline is believed to be dominant for fighting against asthma in practice. Unlike other xanthines, the antiasthmatic effects of doxofylline lack any definite proof of target and mediating mechanism according to previous reports. In this work, the interaction between doxofylline and β2 -AR was investigated by high performance affinity chromatography using frontal analysis and nonlinear model. The methodology involved the immobilization of β2 -AR on the silica gel by a random linking method, the determination of the binding parameters by frontal analysis and nonlinear chromatography and the exploration of the binding mechanism by site-directed molecular docking. The association constant for doxofylline binding to immobilized β2 -AR was determined to be 7.70 × 10(4)  M(-1) by nonlinear chromatography and 5.91 × 10(4)  M(-1) by frontal analysis. Ser(169) and Ser(173) were the binding sites for the receptor-drug interaction on which hydrogen bond was believed to be the main driven force during the interaction. These results indicated that the antiasthmatic effects of doxofylline may be behind the mediating mechanism of β2 -AR. High performance affinity chromatography based on immobilized receptor has potential to become an alternative for drug target confirmation and drug-receptor interaction analysis. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  9. 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.

  10. 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

  11. Sub-lethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis

    Science.gov (United States)

    Kohanski, Michael A.; DePristo, Mark A.; Collins, James J.

    2010-01-01

    Summary Antibiotic resistance arises through mechanisms such as selection of naturally occurring resistant mutants and horizontal gene transfer. Recently, oxidative stress has been implicated as one of the mechanisms whereby bactericidal antibiotics kill bacteria. Here we show that sub-lethal levels of bactericidal antibiotics induce mutagenesis, resulting in heterogeneous increases in the minimum inhibitory concentration for a range of antibiotics, irrespective of the drug target. This increase in mutagenesis correlates with an increase in ROS, and is prevented by the ROS scavenger thiourea and by anaerobic conditions, indicating that sub-lethal concentrations of antibiotics induce mutagenesis by stimulating the production of ROS. We demonstrate that these effects can lead to mutant strains that are sensitive to the applied antibiotic but resistant to other antibiotics. This work establishes a radical-based molecular mechanism whereby sub-lethal levels of antibiotics can lead to multidrug resistance, which has important implications for the widespread use and misuse of antibiotics. PMID:20159551

  12. 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.

  13. In Vitro Activity of Simeprevir against Hepatitis C Virus Genotype 1 Clinical Isolates and Its Correlation with NS3 Sequence and Site-Directed Mutants

    Science.gov (United States)

    Fevery, Bart; Vijgen, Leen; Jacobs, Tom; De Meyer, Sandra; Lenz, Oliver

    2015-01-01

    Simeprevir (TMC435) is a once-daily, single-pill, oral hepatitis C virus (HCV) NS3 protease inhibitor approved for the treatment of chronic HCV infection. Phenotypic characterization of baseline isolates and isolates from HCV genotype 1-infected patients failing with a simeprevir-based regimen was performed using chimeric replicons carrying patient-derived NS3 protease sequences. Cutoff values differentiating between full susceptibility to simeprevir (≤2.0-fold reduction in simeprevir activity) and low-level versus high-level resistance (≥50-fold reduction in simeprevir activity) were determined. The median simeprevir fold change in the 50% effective concentration (FC) of pretreatment genotype 1a isolates, with and without Q80K, and genotype 1b isolates was 11, 0.9, and 0.4, respectively. Naturally occurring NS3 polymorphisms that reduced simeprevir activity, other than Q80K, were uncommon in the simeprevir studies and generally conferred low-level resistance in vitro. Although the proportion of patients with failure differed by HCV geno/subtype and/or presence of baseline Q80K, the level of simeprevir resistance observed at failure was similarly high irrespective of type of failure, HCV genotype 1 subtype, and presence or absence of baseline Q80K. At the end of the study, simeprevir activity against isolates that lost the emerging amino acid substitution returned to pretreatment values. Activity of simeprevir against clinical isolates and site-directed mutant replicons harboring the corresponding single or double amino acid substitutions correlated well, showing that simeprevir resistance can be attributed to these substitutions. In conclusion, pretreatment NS3 isolates were generally fully susceptible (FC, ≤2.0) or conferred low-level resistance to simeprevir in vitro (FC, >2.0 and <50). Treatment failure with a simeprevir-based regimen was associated with emergence of high-level-resistance variants (FC, ≥50). PMID:26392483

  14. 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

  15. 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

    2013-01-01

    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. PMID:23380729

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

    Science.gov (United States)

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

    2007-01-01

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

  17. 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

  18. Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis

    Science.gov (United States)

    Ng, Ashley Shu Mei; Ingham, Philip W.

    2016-01-01

    CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target. Our method provides a streamlined approach to test gRNA efficiency as well as increasing the versatility of conditional gene knock out in zebrafish. PMID:27832146

  19. Ribozyme Mediated gRNA Generation for In Vitro and In Vivo CRISPR/Cas9 Mutagenesis.

    Directory of Open Access Journals (Sweden)

    Raymond Teck Ho Lee

    Full Text Available CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter that was able to induce mutagenesis of its target. Our method provides a streamlined approach to test gRNA efficiency as well as increasing the versatility of conditional gene knock out in zebrafish.

  20. Short Communication

    African Journals Online (AJOL)

    ... et al., 1999). The assay represents a rapid, inexpensive and simple bioassay for testing plant extracts bioactivity which in most cases correlates reasonably well with cytotoxic and anti-tumor properties. The brine shrimp lethality assay is based on the ability of the extract to show lethality in laboratory. Short Communication ...

  1. Short Report

    African Journals Online (AJOL)

    Bioline

    Short Report. Waiting time among acute abdominal emergencies in a Nigerian teaching hospital: causes of delay and consequences. N .Mbah, W. Ek. Opara and N. P. Agwu ... admitted with acute surgical abdomen are most commonly due to financial difficulties. ... private, general and primary health institutions within.

  2. SHORT COMMUNICATION

    African Journals Online (AJOL)

    Tanzania Health Research Bulletin (2005), Vol. 7, No. 2. SHORT COMMUNICATION. Palliative care in Tanzania: a needs assessment study of family caregivers in urban setting. T. NGOMA. Ocean Road Cancer Institute, PO. Box 3592 Dar es Salaam, Tanzania;. Email: ngoma@uccmail.co.tz. Tanzania, with a population of ...

  3. Short communications

    African Journals Online (AJOL)

    2015-05-23

    May 23, 2015 ... the majority of birds in one image (Fig. 2). Figure 2. Partial stack of 100+ Levant Sparrowhawks over the Mara North Conservancy, 23 February 2014. Photo: M. Mockler. Short communications. 33. Figure 1. Photographs confirming the identification of Levant Sparrowhawk. Accipiter brevipes. Photos: M.

  4. Short Comm.

    African Journals Online (AJOL)

    E-LIBRARY

    economic stability. Industrialization according to Britannica Encyclopedia is, “The process of converting to a socio-economic or development in which industry is ... and even secondary schools. Furthermore, disciplines such as Library and Information Science have over the years been. Short Comm. Information Impact:.

  5. Short Communication

    African Journals Online (AJOL)

    Nigerian Journal of Basic and Applied Science (2011), 19(2): 304-307. ISSN 0794-5698. Short Communication. Lead and Cadmium Levels of Five Commonly and Widely Consumed Leafy Vegetables in. Kano State, Nigeria. *M.I. Mohammed and ... the agreed international requirements. This is particularly important for farm ...

  6. Short communication

    African Journals Online (AJOL)

    UPuser

    Short communication. Polymorphisms of the CAST gene in the Meishan and five other pig populations in China. Q.S. Wang. 1. , Y.C. Pan. 1#. , L.B. Sun. 2 and H. Meng. 1. 1 Department of Animal Science, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai. 201101, P.R. China. 2 Shanghai Institute of ...

  7. Effect of dose and dosing rate on the mutagenesis of nitric oxide in ...

    African Journals Online (AJOL)

    Effect of dose and dosing rate on the mutagenesis of nitric oxide in supF shuttle vector. Ji Hye Kim1 and ... Purpose: To determine how the dose and rate of NO• treatment affects mutagenic responses. Methods: Shuttle vector pSP189 was ... form a strong oxidant and nitrating agent, peroxynitrite (ONOO-), which can initiate.

  8. Identification of a novel streptococcal gene cassette mediating SOS mutagenesis in Streptococcus uberis

    NARCIS (Netherlands)

    Varhimo, Emilia; Savijoki, Kirsi; Jalava, Jari; Kuipers, Oscar P.; Varmanen, Pekka

    Streptococci have been considered to lack the classical SOS response, defined by increased mutation after UV exposure and regulation by LexA. Here we report the identification of a potential self-regulated SOS mutagenesis gene cassette in the Streptococcaceae family. Exposure to UV light was found

  9. 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.

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

    Science.gov (United States)

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

    2012-03-01

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

  11. Mutagenesis effects of X-irradiation on the germination, growth and ...

    African Journals Online (AJOL)

    The time of exposure and mutagenesis effects of X-ray on the germination, growth and development of maize (Zea mays) seeds have been investigated employing a nuclear source, (90Sr as X-ray source; 3mA, 70kv). Five sects of zea mays seeds in different packs were irradiated in different times of exposure and later ...

  12. Bromination of deoxycytidine by eosinophil peroxidase: A mechanism for mutagenesis by oxidative damage of nucleotide precursors

    OpenAIRE

    Henderson, Jeffrey P.; Byun, Jaeman; Williams, Michelle V.; McCormick, Michael L.; Parks, William C.; Ridnour, Lisa A.; Heinecke, Jay W.

    2001-01-01

    Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by ...

  13. Pilot study of large-scale production of mutant pigs by ENU mutagenesis

    Science.gov (United States)

    Hai, Tang; Cao, Chunwei; Shang, Haitao; Guo, Weiwei; Mu, Yanshuang; Yang, Shulin; Zhang, Ying; Zheng, Qiantao; Zhang, Tao; Wang, Xianlong; Liu, Yu; Kong, Qingran; Li, Kui; Wang, Dayu; Qi, Meng; Hong, Qianlong; Zhang, Rui; Wang, Xiupeng; Jia, Qitao; Wang, Xiao; Qin, Guosong; Li, Yongshun; Luo, Ailing; Jin, Weiwu; Yao, Jing; Huang, Jiaojiao; Zhang, Hongyong; Li, Menghua; Xie, Xiangmo; Zheng, Xuejuan; Guo, Kenan; Wang, Qinghua; Zhang, Shibin; Li, Liang; Xie, Fei; Zhang, Yu; Weng, Xiaogang; Yin, Zhi; Hu, Kui; Cong, Yimei; Zheng, Peng; Zou, Hailong; Xin, Leilei; Xia, Jihan; Ruan, Jinxue; Li, Hegang; Zhao, Weiming; Yuan, Jing; Liu, Zizhan; Gu, Weiwang; Li, Ming; Wang, Yong; Wang, Hongmei; Yang, Shiming; Liu, Zhonghua; Wei, Hong; Zhao, Jianguo; Zhou, Qi; Meng, Anming

    2017-01-01

    N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research. DOI: http://dx.doi.org/10.7554/eLife.26248.001 PMID:28639938

  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. 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…

  16. Short Stature

    DEFF Research Database (Denmark)

    Christesen, Henrik Boye Thybo; Pedersen, Birgitte Tønnes; Pournara, Effie

    2016-01-01

    -scale, non-interventional, multinational study. The patient cohort consisted of 5996 short pediatric patients diagnosed with growth hormone deficiency (GHD), Turner syndrome (TS) or born small for gestational age (SGA). The proportions of children with baseline height standard deviation score (SDS) below...... clinical cut-off values (-2 SDS for GHD and TS; -2.5 SDS for SGA) based on national growth references and WHO growth standards/references were compared for children aged ....0001), TS (21%; PSGA (32%; P

  17. In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model.

    Science.gov (United States)

    Kim, H J; Nishikawa, S; Tokutomi, Y; Takenaka, H; Hamada, M; Kuby, S A; Uesugi, S

    1990-02-06

    Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP2- and for AMP2- in human cytosolic adenylate kinase (EC 2.7.4.3, hAK1), we have investigated the enzymic effects of replacement of the arginine residues (R44, R132, R138, and R149), which had been assumed by Pai et al. [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45] to interact with the phosphoryl groups of AMP2- and MgATP2-. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 [Kim, H. J., Nishikawa, S., Tanaka, T., Uesugi, S., Takenaka, H., Hamada, M., & Kuby, S. A. (1989) Protein Eng. 2, 379-386] to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the Km,app values for AMP2- of the mutant enzymes, the relatively small increases in the Km,app values for MgATP2-, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. (1977) have been reversed and that their ATP-binding site may be assigned as the AMP site.

  18. 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.

  19. 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.

  20. Short esophagus.

    Science.gov (United States)

    Kunio, Nicholas R; Dolan, James P; Hunter, John G

    2015-06-01

    In the presence of long-standing and severe gastroesophageal reflux disease, patients can develop various complications, including a shortened esophagus. Standard preoperative testing in these patients should include endoscopy, esophagography, and manometry, whereas the objective diagnosis of a short esophagus must be made intraoperatively following adequate mediastinal mobilization. If left untreated, it is a contributing factor to the high recurrence rate following fundoplications or repair of large hiatal hernias. A laparoscopic Collis gastroplasty combined with an antireflux procedure offers safe and effective therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line.

    Science.gov (United States)

    Chu, Van Trung; Graf, Robin; Wirtz, Tristan; Weber, Timm; Favret, Jeremy; Li, Xun; Petsch, Kerstin; Tran, Ngoc Tung; Sieweke, Michael H; Berek, Claudia; Kühn, Ralf; Rajewsky, Klaus

    2016-11-01

    Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation. This screening system does not require deep sequencing and may serve as a precedent for the application of CRISPR/Cas9 to primary mouse cells.

  2. Advances in Radiation Mutagenesis through Studies on Drosophila

    Science.gov (United States)

    Muller, H. J.

    1958-06-01

    The approximately linear relation between radiation dose and induced lethals known for Drosophila spermatozoa, is now extended to spermatids. Data are included regarding oogonia. The linearity principle has been confined for minute structural changes in sperm as multi-hit events, on about the 1.5 power of the dose, long known for spermatozoa, is now extended to spermatids and late oocytes, for relatively short exposures. are found to allow union of broken chromosomes. Therefore, the frequencies are lower for more dispersed exposures of varies with lethals induced in late oocytes follow the same frequency pattern and there fore are multi-hit events. Yet han spermatozoan irradiation that two broken ends derived from nonreciprocal. The following is the order of decreasing radiation mutability of different stages found by ourselves and others: spermatids, spermatozoa in females, spermatozoa 0 to 1 day before ejaculation, earlier spermatozoa, late oocytes, gonia of either sex. Lethal frequencies for these stages range over approximately an order of magnitude, gross structural changes far more widely. Of potential usefulness is our extension of genesis by anoxia, known for spermatozoa in adult males, to those in pupal males and in females, to sperion is especially marked but the increase caused by substituting oxygen for air is less marked, perhaps because of enzymatic differences. In contrast, the induction of gross structural changes in oocytes, but not in spermatids, is markedly reduced by oxygen post-treatment; it is increased by dehydration. The efficacy of induction of structural changes by treatment of spermatozoa, whether with radiation or chemical mutagen, is correlated with the conditions of sperm utilization and egg production. Improving our perspective on radiation effects, some 800,000 offspring have been scored for spontaneous visible mutations of 13 specific loci. The average point-mutation rate was 0.5 to 1.0 per locus among 10/sup 5/ germ cells. Most

  3. Transposon Tn5 mutagenesis of pseudomonas fluorescens to isolate mutants deficient in antibacterial activity.

    Science.gov (United States)

    Rajendran, N; Jahn, D; Jayaraman, K; Marahiel, M A

    1994-01-15

    Pseudomonas fluorescens was subjected to insertion mutagenesis studies using the transposon Tn5-GM to generate mutants deficient in antibacterial activity minus mutants. The transposon located on the temperature-sensitive plasmid pCHR84 was conjugally transferred into the non-pathogenic pseudomonad using the triparental mating procedure. Random integration of Tn5-GM into the chromosome of P. fluorescens was achieved by heat treatment of the transformed cells at 42 degrees C. Approximately 2% of transconjugants revealed an auxotrophic phenotype indicating efficient integration of the employed transposon into the chromosome of P. fluorescens. One transposon insertion mutant was obtained showing an antibacterial activity minus phenotype. This mutant (MM-7) was found to be defective in the production of an unidentified antibacterial compound against B. subtilis. These results introduce Tn5 transposon mutagenesis as a new useful tool for the molecular analysis of P. fluorescens.

  4. Prediction of enzyme mutant activity using computational mutagenesis and incremental transduction.

    Science.gov (United States)

    Basit, Nada; Wechsler, Harry

    2011-01-01

    Wet laboratory mutagenesis to determine enzyme activity changes is expensive and time consuming. This paper expands on standard one-shot learning by proposing an incremental transductive method (T2bRF) for the prediction of enzyme mutant activity during mutagenesis using Delaunay tessellation and 4-body statistical potentials for representation. Incremental learning is in tune with both eScience and actual experimentation, as it accounts for cumulative annotation effects of enzyme mutant activity over time. The experimental results reported, using cross-validation, show that overall the incremental transductive method proposed, using random forest as base classifier, yields better results compared to one-shot learning methods. T2bRF is shown to yield 90% on T4 and LAC (and 86% on HIV-1). This is significantly better than state-of-the-art competing methods, whose performance yield is at 80% or less using the same datasets.

  5. Prediction of Enzyme Mutant Activity Using Computational Mutagenesis and Incremental Transduction

    Directory of Open Access Journals (Sweden)

    Nada Basit

    2011-01-01

    Full Text Available Wet laboratory mutagenesis to determine enzyme activity changes is expensive and time consuming. This paper expands on standard one-shot learning by proposing an incremental transductive method (T2bRF for the prediction of enzyme mutant activity during mutagenesis using Delaunay tessellation and 4-body statistical potentials for representation. Incremental learning is in tune with both eScience and actual experimentation, as it accounts for cumulative annotation effects of enzyme mutant activity over time. The experimental results reported, using cross-validation, show that overall the incremental transductive method proposed, using random forest as base classifier, yields better results compared to one-shot learning methods. T2bRF is shown to yield 90% on T4 and LAC (and 86% on HIV-1. This is significantly better than state-of-the-art competing methods, whose performance yield is at 80% or less using the same datasets.

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

    Science.gov (United States)

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

    2015-11-01

    A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ~3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Transposon mutagenesis identifies genes that cooperate with mutant Pten in breast cancer progression

    Science.gov (United States)

    Rangel, Roberto; Lee, Song-Choon; Hon-Kim Ban, Kenneth; Guzman-Rojas, Liliana; Mann, Michael B.; Newberg, Justin Y.; McNoe, Leslie A.; Selvanesan, Luxmanan; Ward, Jerrold M.; Rust, Alistair G.; Chin, Kuan-Yew; Black, Michael A.; Jenkins, Nancy A.; Copeland, Neal G.

    2016-01-01

    Triple-negative breast cancer (TNBC) has the worst prognosis of any breast cancer subtype. To better understand the genetic forces driving TNBC, we performed a transposon mutagenesis screen in a phosphatase and tensin homolog (Pten) mutant mice and identified 12 candidate trunk drivers and a much larger number of progression genes. Validation studies identified eight TNBC tumor suppressor genes, including the GATA-like transcriptional repressor TRPS1. Down-regulation of TRPS1 in TNBC cells promoted epithelial-to-mesenchymal transition (EMT) by deregulating multiple EMT pathway genes, in addition to increasing the expression of SERPINE1 and SERPINB2 and the subsequent migration, invasion, and metastasis of tumor cells. Transposon mutagenesis has thus provided a better understanding of the genetic forces driving TNBC and discovered genes with potential clinical importance in TNBC. PMID:27849608

  8. Back to BAC: The Use of Infectious Clone Technologies for Viral Mutagenesis

    Directory of Open Access Journals (Sweden)

    Robyn N. Hall

    2012-02-01

    Full Text Available Bacterial artificial chromosome (BAC vectors were first developed to facilitate the propagation and manipulation of large DNA fragments in molecular biology studies for uses such as genome sequencing projects and genetic disease models. To facilitate these studies, methodologies have been developed to introduce specific mutations that can be directly applied to the mutagenesis of infectious clones (icBAC using BAC technologies. This has resulted in rapid identification of gene function and expression at unprecedented rates. Here we review the major developments in BAC mutagenesis in vitro. This review summarises the technologies used to construct and introduce mutations into herpesvirus icBAC. It also explores developing technologies likely to provide the next leap in understanding these important viruses.

  9. Cell-mediated mutagenesis and cell transformation of mammalian cells by chemical carcinogens. [Rats, hamsters

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. Model of SOS-induced mutagenesis in bacteria Escherichia coli under ultraviolet irradiation.

    Science.gov (United States)

    Belov, Oleg V; Krasavin, Evgeny A; Parkhomenko, Alexander Yu

    2009-12-07

    A mathematical model of the mutation process in bacteria Escherichia coli induced by ultraviolet radiation is developed. Our model is based on the experimental data characterizing the main processes of the bacterial SOS response. Here we have modeled a whole sequence of the events leading to the fixation of the primary DNA lesion as a point mutation. A quantitative analysis of the key ways of the SOS mutagenesis was performed in terms of modern system biology. The dynamic changes of the basic SOS protein concentrations and the process of the translesion synthesis by the modified replication complex are described quantitatively. We have also demonstrated the applicability of the developed model to the description of the mutagenesis in individual genes. As an example, an estimation of the mutation frequency in E. coli's lacI gene is performed.

  11. 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.

  12. Bromination of deoxycytidine by eosinophil peroxidase: A mechanism for mutagenesis by oxidative damage of nucleotide precursors

    Science.gov (United States)

    Henderson, Jeffrey P.; Byun, Jaeman; Williams, Michelle V.; McCormick, Michael L.; Parks, William C.; Ridnour, Lisa A.; Heinecke, Jay W.

    2001-01-01

    Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by eosinophil peroxidase was taken up by cultured cells and incorporated into genomic DNA as 5-bromodeoxyuridine. Although previous studies have focused on oxidation of chromosomal DNA, our observations suggest another mechanism for oxidative damage of DNA. In this scenario, peroxidase-catalyzed halogenation of nucleotide precursors yields products that subsequently can be incorporated into DNA. Because the thymine analog 5-BrUra mispairs with guanine in DNA, generation of brominated pyrimidines by eosinophils might constitute a mechanism for cytotoxicity and mutagenesis at sites of inflammation. PMID:11172002

  13. 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.

  14. Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome

    OpenAIRE

    Messerle, Martin; Crnkovic, Irena; Hammerschmidt, Wolfgang; Ziegler, Heike; Koszinowski, Ulrich H

    1997-01-01

    A strategy for cloning and mutagenesis of an infectious herpesvirus genome is described. The mouse cytomegalovirus genome was cloned and maintained as a 230 kb bacterial artificial chromosome (BAC) in E. coli. Transfection of the BAC plasmid into eukaryotic cells led to a productive virus infection. The feasibility to introduce targeted mutations into the BAC cloned virus genome was shown by mutation of the immediate-early 1 gene and generation of a mutant virus. Thus, the complete constructi...

  15. Prevention of mutagenesis: new potential mechanisms of metformin action in neoplastic cells.

    Science.gov (United States)

    Bost, Frédéric; Ben-Sahra, Issam; Tanti, Jean-François

    2012-04-01

    Several experimental and epidemiologic studies have shown that the antidiabetes drug metformin has antitumor properties. The report by Algire and colleagues in this issue of the journal (beginning on page 536) shows for the first time that metformin reduces mutagenesis induced by reactive oxygen species. This report offers new perspectives on metformin in cancer prevention and provides a new mechanism for the reduction of cancer risk in diabetic patients treated with this drug. 2012 AACR

  16. General Mutagenesis of F Plasmid TraI Reveals Its Role in Conjugative Regulation

    OpenAIRE

    Haft, Rembrandt J. F.; Palacios, Gilberto; Nguyen, Tran; Mally, Manuela; Gachelet, Eliora G.; Zechner, Ellen L.; Traxler, Beth

    2006-01-01

    Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene traI using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI...

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

    OpenAIRE

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

    2008-01-01

    Controversy regarding genetically modified (GM) plants and their potential impact on human health contrasts with the tacit acceptance of other plants that were also modified, but not considered as GM products (e.g., varieties raised through conventional breeding such as mutagenesis). What is beyond the phenotype of these improved plants? Should mutagenized plants be treated differently from transgenics? We have evaluated the extent of transcriptome modification occurring ...

  18. A plasmid-transposon hybrid mutagenesis system effective in a broad range of Enterobacteria

    Directory of Open Access Journals (Sweden)

    Rita eMonson

    2015-12-01

    Full Text Available Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.

  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. mtDNA Mutagenesis Disrupts Pluripotent Stem Cell Function by Altering Redox Signaling

    Directory of Open Access Journals (Sweden)

    Riikka H. Hämäläinen

    2015-06-01

    Full Text Available mtDNA mutagenesis in somatic stem cells leads to their dysfunction and to progeria in mouse. The mechanism was proposed to involve modification of reactive oxygen species (ROS/redox signaling. We studied the effect of mtDNA mutagenesis on reprogramming and stemness of pluripotent stem cells (PSCs and show that PSCs select against specific mtDNA mutations, mimicking germline and promoting mtDNA integrity despite their glycolytic metabolism. Furthermore, mtDNA mutagenesis is associated with an increase in mitochondrial H2O2, reduced PSC reprogramming efficiency, and self-renewal. Mitochondria-targeted ubiquinone, MitoQ, and N-acetyl-L-cysteine efficiently rescued these defects, indicating that both reprogramming efficiency and stemness are modified by mitochondrial ROS. The redox sensitivity, however, rendered PSCs and especially neural stem cells sensitive to MitoQ toxicity. Our results imply that stem cell compartment warrants special attention when the safety of new antioxidants is assessed and point to an essential role for mitochondrial redox signaling in maintaining normal stem cell function.

  1. 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

    2012-12-01

    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.

  2. Ionizing radiation-induced bystander mutagenesis and adaptation: Quantitative and temporal aspects

    Science.gov (United States)

    Zhang, Ying; Zhou, Junqing; Baldwin, Joseph; Held, Kathryn D; Prise, Kevin M; Redmond, Robert W.; Liber, Howard L.

    2009-01-01

    This work explores several quantitative aspects of radiation-induced bystander mutagenesis in WTK1 human lymphoblast cells. Gamma-irradiation of cells was used to generate conditioned medium containing bystander signals, and that medium was transferred onto naïve recipient cells. Kinetic studies revealed that it required up to one hour to generate sufficient signal to induce the maximal level of mutations at the thymidine kinase locus in the bystander cells receiving the conditioned medium. Furthermore, it required at least one hour of exposure to the signal in the bystander cells to induce mutations. Bystander signal was fairly stable in the medium, requiring 12–24 hours to diminish. Medium that contained bystander signal was rendered ineffective by a 4-fold dilution; in contrast a greater than 20-fold decrease in the cell number irradiated to generate a bystander signal was needed to eliminate bystander-induced mutagenesis. This suggested some sort of feedback inhibition by bystander signal that prevented the signaling cells from releasing more signal. Finally, an ionizing radiation-induced adaptive response was shown to be effective in reducing bystander mutagenesis; in addition, low levels of exposure to bystander signal in the transferred medium induced adaptation that was effective in reducing mutations induced by subsequent γ-ray exposures. PMID:19695271

  3. Ionizing radiation-induced bystander mutagenesis and adaptation: Quantitative and temporal aspects

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Ying; Zhou Junqing; Baldwin, Joseph [Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO (United States); Held, Kathryn D. [Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA (United States); Prise, Kevin M. [Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Belfast (United Kingdom); Redmond, Robert W. [Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA (United States); Liber, Howard L., E-mail: howard.liber@colostate.edu [Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO (United States); University of Colorado Cancer Center, Denver, CO (United States)

    2009-12-01

    This work explores several quantitative aspects of radiation-induced bystander mutagenesis in WTK1 human lymphoblast cells. Gamma-irradiation of cells was used to generate conditioned medium containing bystander signals, and that medium was transferred onto naive recipient cells. Kinetic studies revealed that it required up to 1 h to generate sufficient signal to induce the maximal level of mutations at the thymidine kinase locus in the bystander cells receiving the conditioned medium. Furthermore, it required at least 1 h of exposure to the signal in the bystander cells to induce mutations. Bystander signal was fairly stable in the medium, requiring 12-24 h to diminish. Medium that contained bystander signal was rendered ineffective by a 4-fold dilution; in contrast a greater than 20-fold decrease in the cell number irradiated to generate a bystander signal was needed to eliminate bystander-induced mutagenesis. This suggested some sort of feedback inhibition by bystander signal that prevented the signaling cells from releasing more signal. Finally, an ionizing radiation-induced adaptive response was shown to be effective in reducing bystander mutagenesis; in addition, low levels of exposure to bystander signal in the transferred medium induced adaptation that was effective in reducing mutations induced by subsequent {gamma}-ray exposures.

  4. 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.

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

    Science.gov (United States)

    Aubert, Martine; Boyle, Nicole M; Stone, Daniel; Stensland, Laurence; Huang, Meei-Li; Magaret, Amalia S; Galetto, Roman; Rawlings, David J; Scharenberg, Andrew M; Jerome, Keith R

    2014-02-04

    Following acute infection, herpes simplex virus (HSV) establishes latency in sensory neurons, from which it can reactivate and cause recurrent disease. Available antiviral therapies do not affect latent viral genomes; therefore, they do not prevent reactivation following therapy cessation. One possible curative approach involves the introduction of DNA double strand breaks in latent HSV genomes by rare-cutting endonucleases, leading to mutagenesis of essential viral genes. We tested this approach in an in vitro HSV latency model using the engineered homing endonuclease (HE) HSV1m5, which recognizes a sequence in the HSV-1 gene UL19, encoding the virion protein VP5. Coexpression of the 3'-exonuclease Trex2 with HEs increased HE-mediated mutagenesis frequencies up to sixfold. Following HSV1m5/Trex2 delivery with adeno-associated viral (AAV) vectors, the target site was mutated in latent HSV genomes with no detectable cell toxicity. Importantly, HSV production by latently infected cells after reactivation was decreased after HSV1m5/Trex2 exposure. Exposure to histone deacetylase inhibitors prior to HSV1m5/Trex2 treatment increased mutagenesis frequencies of latent HSV genomes another two- to fivefold, suggesting that chromatin modification may be a useful adjunct to gene-targeting approaches. These results support the continuing development of HEs and other nucleases (ZFNs, TALENs, CRISPRs) for cure of chronic viral infections.Molecular Therapy-Nucleic Acids (2014) 3, e1; doi:10.1038/mtna.2013.75; published online 4 February 2014.

  6. 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

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

    Directory of Open Access Journals (Sweden)

    Moskowitz Ivan

    2009-03-01

    Full Text Available Abstract Background ENU-mutagenesis is a powerful technique to identify genes regulating mammalian development. To functionally annotate the distal region of mouse chromosome 4, we performed an ENU-mutagenesis screen using a balancer chromosome targeted to this region of the genome. Results We isolated 11 lethal lines that map to the region of chromosome 4 between D4Mit117 and D4Mit281. These lines form 10 complementation groups. The majority of lines die during embryonic development between E5.5 and E12.5 and display defects in gastrulation, cardiac development, and craniofacial development. One line displayed postnatal lethality and neurological defects, including ataxia and seizures. Conclusion These eleven mutants allow us to query gene function within the distal region of mouse chromosome 4 and demonstrate that new mouse models of mammalian developmental defects can easily and quickly be generated and mapped with the use of ENU-mutagenesis in combination with balancer chromosomes. The low number of mutations isolated in this screen compared with other balancer chromosome screens indicates that the functions of genes in different regions of the genome vary widely.

  8. 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.

  9. 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.

  10. Tissue-specific mutagenesis by N-butyl-N-(4-hydroxybutyl)nitrosamine as the basis for urothelial carcinogenesis.

    Science.gov (United States)

    He, Zhiming; Kosinska, Wieslawa; Zhao, Zhong-Lin; Wu, Xue-Ru; Guttenplan, Joseph B

    2012-02-18

    Bladder cancer is one of the few cancers that have been linked to carcinogens in the environment and tobacco smoke. Of the carcinogens tested in mouse chemical carcinogenesis models, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) is one that reproducibly causes high-grade, invasive cancers in the urinary bladder, but not in any other tissues. However, the basis for such a high-level tissue-specificity has not been explored. Using mutagenesis in lacI (Big Blue™) mice, we show here that BBN is a potent mutagen and it causes high-level of mutagenesis specifically in the epithelial cells (urothelial) of the urinary bladder. After a 2-6-week treatment of 0.05% BBN in the drinking water, mutagenesis in urothelial cells of male and female mice was about two orders of magnitude greater than the spontaneous mutation background. In contrast, mutagenesis in smooth muscle cells of the urinary bladder was about five times lower than in urothelial tissue. No appreciable increase in mutagenesis was observed in kidney, ureter, liver or forestomach. In lacI (Big Blue™) rats, BBN mutagenesis was also elevated in urothelial cells, albeit not nearly as profoundly as in mice. This provides a potential explanation as to why rats are less prone than mice to the formation of aggressive form of bladder cancer induced by BBN. Our results suggest that the propensity to BBN-triggered mutagenesis of urothelial cells underlies its heightened susceptibility to this carcinogen and that mutagenesis induced by BBN represents a novel model for initiation of bladder carcinogenesis. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. [IXR1 and HMO1 genes jointly control the level of spontaneous mutagenesis in yeast Saccharomyces cerevisiae].

    Science.gov (United States)

    Fedorov, D V; Koval'tsova, S V; Peshekhonov, V T; Korolev, V G

    2010-06-01

    The yeast genes IXR1 and HMO1 encode proteins belonging to the family of chromatin nonhistone proteins, which are able to recognize and bind to irregular DNA structures. The full deletion of gene IXR1 leads to an increase in cell resistance to the lethal action of UV light, gamma-rays, and MMS, increases spontaneous mutagenesis and significantlly decreases the level of UV-induced mutations. It was earlier demonstrated in our works that the hmo 1 mutation renders cells sensitive to the lethal action of cisplatin and virtually does not affect the sensitivity to UV light. Characteristically, the rates of spontaneous and UV-induced mutagenesis in the mutant are increased. Epistatic analysis of the double mutation hmo 1 ixr1 demonstrated that the interaction of these genes in relation to the lethal effect of cisplatin and UV light, as well as UV-induced mutagenesis, is additive. This suggests that the products of genes HMO1 and IXR1 participate in different repair pathways. The ixr1 mutation significantly increases the rate of spontaneous mutagenesis mediated by replication errors, whereas mutation hmo 1 increases the rate of repair mutagenesis. In wild-type cells, the level of spontaneous mutagenesis was nearly one order of magnitude lower than that obtained in cells of the double mutant. Consequently, the combined activity of the Hmo 1 and the Ixr1 proteins provides efficient correction of both repair and replication errors.

  12. Novel bifunctional DATA chelator for quick access to site-directed PET 68Ga-radiotracers: preclinical proof-of-principle with [Tyr3]octreotide.

    Science.gov (United States)

    Nock, Berthold A; Kaloudi, Aikaterini; Nagel, Johannes; Sinnes, Jean-Philippe; Roesch, Frank; Maina, Theodosia

    2017-10-31

    Molecular imaging of tumors with the PET radionuclide 68Ga has gained momentum in clinical oncology due to the expanding availability of commercial 68Ge/68Ga-generators in combination with state-of-the-art PET/CT and PET/MRI hybrid imaging systems. Concurrently, interesting peptide-based or small-size vectors have been developed for theranostic use in cancer patients. Owing to the short half-life of 68Ga (t1/2 = 67.7 min) and the sensitivity of many targeting biomolecules, labeling and kit reconstitution in mild conditions allowing for quick access to ready-for-injection PET-tracers are highly desirable. The novel DATA ((6-pentanoic acid)-6-(amino)methy-1,4-diazepinetriacetate) chelator previously showing promising qualities for kit type labeling, was coupled to TOC ([Tyr3]octreotide). We herein report results from a first proof-of-principle study directly comparing 67Ga-DATA-TOC with the well-established 67Ga-DOTA-TOC in a series of preclinical models. Both analogs were shown to be sst2-preferring and specifically internalized in AR42J and HEK293-hsst2 cells, with 67Ga-DOTA-TOC internalizing faster in both cell lines. Similarly, after injection in mice bearing either AR42J or HEK293-hsst2 tumors, both tracers efficiently and specifically localized in the implants. Whereas 67Ga-DOTA-TOC exhibited higher tumor values, 67Ga-DATA-TOC cleared faster from background tissues. These findings support the suitability of the newly introduced bifunctional chelator DATA as a reliable, quick and convenient means for labeling medically relevant vectors with the PET radiometal 68Ga.

  13. Modeling insertional mutagenesis using gene length and expression in murine embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Alex S Nord

    2007-07-01

    Full Text Available High-throughput mutagenesis of the mammalian genome is a powerful means to facilitate analysis of gene function. Gene trapping in embryonic stem cells (ESCs is the most widely used form of insertional mutagenesis in mammals. However, the rules governing its efficiency are not fully understood, and the effects of vector design on the likelihood of gene-trapping events have not been tested on a genome-wide scale.In this study, we used public gene-trap data to model gene-trap likelihood. Using the association of gene length and gene expression with gene-trap likelihood, we constructed spline-based regression models that characterize which genes are susceptible and which genes are resistant to gene-trapping techniques. We report results for three classes of gene-trap vectors, showing that both length and expression are significant determinants of trap likelihood for all vectors. Using our models, we also quantitatively identified hotspots of gene-trap activity, which represent loci where the high likelihood of vector insertion is controlled by factors other than length and expression. These formalized statistical models describe a high proportion of the variance in the likelihood of a gene being trapped by expression-dependent vectors and a lower, but still significant, proportion of the variance for vectors that are predicted to be independent of endogenous gene expression.The findings of significant expression and length effects reported here further the understanding of the determinants of vector insertion. Results from this analysis can be applied to help identify other important determinants of this important biological phenomenon and could assist planning of large-scale mutagenesis efforts.

  14. 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.

  15. 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.

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

    Science.gov (United States)

    Patel, Jinesh D; Wright, Robert J; Auld, Dick; Chandnani, Rahul; Goff, Valorie H; Ingles, Jennifer; Pierce, Gary J; Torres, Manuel J; Paterson, Andrew H

    2014-04-01

    Genetic improvements for many fiber traits are obtained by mutagenesis of elite cottons, mitigating genetic uniformity in this inbred polyploid by contributing novel alleles important to ongoing crop improvement. The elite gene pool of cotton (Gossypium spp.) has less diversity than those of most other major crops, making identification of novel alleles important to ongoing crop improvement. A total of 3,164 M5 lines resulting from ethyl methanesulfonate (EMS) mutagenesis of two G. hirsutum breeding lines, TAM 94L-25 and Acala 1517-99, were characterized for basic components of fiber quality and selected yield components. Across all measured traits, the ranges of phenotypic values among the mutant lines were consistently larger than could be explained by chance (5.27-10.1 for TAM 94 L-25 and 5.29-7.94 standard deviations for Acala 1517-99-derived lines). Multi-year replicated studies confirmed a genetic basis for these differences, showing significant correlations between lines across years and environments. A subset of 157 lines selected for superior fiber qualities, including fiber elongation (22 lines), length (22), lint percent (17), fineness (23), Rd value (21), strength (19), uniformity (21) and multiple attributes in a selection index (26) were compared to 55 control lines in replicated trials in both Texas and Georgia. For all traits, mutant lines showing substantial and statistically significant improvements over control lines were found, in most cases from each of the two genetic backgrounds. This indicates that genetic improvements for a wide range of fiber traits may be obtained from mutagenesis of elite cottons. Indeed, lines selected for one fiber trait sometimes conferred additional attributes, suggesting pleiotropic effects of some mutations and offering multiple benefits for the incorporation of some alleles into mainstream breeding programs.

  17. Mechanism of DNA alkylation-induced transcriptional stalling, lesion bypass, and mutagenesis.

    Science.gov (United States)

    Xu, Liang; Wang, Wei; Wu, Jiabin; Shin, Ji Hyun; Wang, Pengcheng; Unarta, Ilona Christy; Chong, Jenny; Wang, Yinsheng; Wang, Dong

    2017-08-22

    Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, interfere with the efficiency and accuracy of DNA replication and transcription. However, the molecular mechanisms of DNA alkylation-induced transcriptional stalling and mutagenesis remain unknown. In this study, we systematically investigated how RNA polymerase II (pol II) recognizes and bypasses regioisomeric O 2 -, N 3-, and O 4 -ethylthymidine ( O 2 -, N 3-, and O 4 -EtdT) lesions. We observed distinct pol II stalling profiles for the three regioisomeric EtdT lesions. Intriguingly, pol II stalling at O 2 -EtdT and N 3-EtdT sites is exacerbated by TFIIS-stimulated proofreading activity. Assessment for the impact of the EtdT lesions on individual fidelity checkpoints provided further mechanistic insights, where the transcriptional lesion bypass routes for the three EtdT lesions are controlled by distinct fidelity checkpoints. The error-free transcriptional lesion bypass route is strongly favored for the minor-groove O 2 -EtdT lesion. In contrast, a dominant error-prone route stemming from GMP misincorporation was observed for the major-groove O 4 -EtdT lesion. For the N 3-EtdT lesion that disrupts base pairing, multiple transcriptional lesion bypass routes were found. Importantly, the results from the present in vitro transcriptional studies are well correlated with in vivo transcriptional mutagenesis analysis. Finally, we identified a minor-groove-sensing motif from pol II (termed Pro-Gate loop). The Pro-Gate loop faces toward the minor groove of RNA:DNA hybrid and is involved in modulating the translocation of minor-groove alkylated DNA template after nucleotide incorporation opposite the lesion. Taken together, this work provides important mechanistic insights into transcriptional stalling, lesion bypass, and mutagenesis of alkylated DNA lesions.

  18. 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.

  19. Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis

    Directory of Open Access Journals (Sweden)

    Kathryn E. Tiller

    2017-09-01

    Full Text Available The identification of mutations that enhance antibody affinity while maintaining high antibody specificity and stability is a time-consuming and laborious process. Here, we report an efficient methodology for systematically and rapidly enhancing the affinity of antibody variable domains while maximizing specificity and stability using novel synthetic antibody libraries. Our approach first uses computational and experimental alanine scanning mutagenesis to identify sites in the complementarity-determining regions (CDRs that are permissive to mutagenesis while maintaining antigen binding. Next, we mutagenize the most permissive CDR positions using degenerate codons to encode wild-type residues and a small number of the most frequently occurring residues at each CDR position based on natural antibody diversity. This mutagenesis approach results in antibody libraries with variants that have a wide range of numbers of CDR mutations, including antibody domains with single mutations and others with tens of mutations. Finally, we sort the modest size libraries (~10 million variants displayed on the surface of yeast to identify CDR mutations with the greatest increases in affinity. Importantly, we find that single-domain (VHH antibodies specific for the α-synuclein protein (whose aggregation is associated with Parkinson’s disease with the greatest gains in affinity (>5-fold have several (four to six CDR mutations. This finding highlights the importance of sampling combinations of CDR mutations during the first step of affinity maturation to maximize the efficiency of the process. Interestingly, we find that some natural diversity mutations simultaneously enhance all three key antibody properties (affinity, specificity, and stability while other mutations enhance some of these properties (e.g., increased specificity and display trade-offs in others (e.g., reduced affinity and/or stability. Computational modeling reveals that improvements in affinity

  20. Induction of apomixis and fixation of heterosis in Egyptian rice Hybrid1 line using colchicine mutagenesis

    OpenAIRE

    Reda M. Gaafar; Adel R. El Shanshoury; Ahmad A. El Hisseiwy; Mahmoud A. AbdAlhak; Aimn F. Omar; Mohammad M. Abd El Wahab; Randa S. Nofal

    2017-01-01

    It is known that hybrid rice yields 15–20% over inbred varieties in first generation because of heterosis. However, heterosis is normally broken due to segregation. Applying apomixis produces plants as a clone of mother plant and overcomes the problem of breaking heterosis. In order to fix heterosis in the Egyptian rice Hybrid1, their seeds were mutagenized in 0.2% colchicine for two time periods 24 and 50 h. After colchicine mutagenesis, rice seedlings were grown in the field till maturation...

  1. Mutations within Four Distinct Gag Proteins Are Required To Restore Replication of Human Immunodeficiency Virus Type 1 after Deletion Mutagenesis within the Dimerization Initiation Site

    Science.gov (United States)

    Liang, Chen; Rong, Liwei; Quan, Yudong; Laughrea, Michael; Kleiman, Lawrence; Wainberg, Mark A.

    1999-01-01

    Human immunodeficiency virus type 1 (HIV-1) genomic RNA segments at nucleotide (nt) positions +240 to +274 are thought to form a stem-loop secondary structure, termed SL1, that serves as a dimerization initiation site for viral genomic RNA. We have generated two distinct deletion mutations within this region, termed BH10-LD3 and BH10-LD4, involving nt positions +238 to +253 and +261 to +274, respectively, and have shown that each of these resulted in significant diminutions in levels of viral infectiousness. However, long-term culture of each of these viruses in MT-2 cells resulted in a restoration of infectiousness, due to a series of compensatory point mutations within four distinct proteins that are normally cleaved from the Gag precursor. In the case of BH10-LD3, these four mutations were MA1, CA1, MP2, and MNC, and they involved changes of amino acid Val-35 to Ile within the matrix protein (MA), Ile-91 to Thr within the capsid (CA), Thr-12 to Ile within p2, and Thr-24 to Ile within the nucleocapsid (NC). The order in which these mutations were acquired by the mutated BH10-LD3 was MNC > CA1 > MP2 > MA1. The results of site-directed mutagenesis studies confirmed that each of these four substitutions contributed to the increased viability of the mutated BH10-LD3 viruses and that the MNC substitution, which was acquired first, played the most important role in this regard. Three point mutations, MP2, MNC, and MA2, were also shown to be sequentially acquired by viruses that had emerged in culture from the BH10-LD4 deletion. The first two of these were identical to those described above, while the last involved a change of Val-35 to Leu. All three of these substitutions were necessary to restore the infectiousness of mutated BH10-LD4 viruses to wild-type levels, although the MP2 mutation alone, but neither of the other two substitutions, was able to confer some viability on BH10-LD4 viruses. Studies of viral RNA packaging showed that the BH10-LD4 deletion only

  2. Molecular evolution of Fome lignosus laccase by ethyl methane sulfonate-based random mutagenesis in vitro.

    Science.gov (United States)

    Hu, Mei-Rong; Chao, Ya-Peng; Zhang, Guo-Qing; Yang, Xiu-Qing; Xue, Zhi-Quan; Qian, Shi-Jun

    2007-12-01

    In order to improve the laccase activity, mutant libraries are constructed through ethyl methane sulfonate-based (EMS) random mutagenesis. Mutagenesis improved expression 3.7-fold to 144 mgl(-1) laccase in yeast, together with a 1.4-fold increase in K(cat). Thus, the total activity is enhanced 5-fold for 2,2'-azino-bis 3-ethylbenzothiaoline-6-sulfonic acid (ABTS). In the presence of 0.6mM copper, the highest activity value reached 30 Uml(-1) after a 3-day cultivation at a temperature of 30 degrees C(.) In comparison with the wild type, the best mutant enzymatic properties (K(m) for ABTS and guaiacol, thermo- and pH stability, optimal pH) are not changed. Moreover, amino acid sequence analysis indicates that there are four substitutions in the best mutant laccase (Gly160Asp, Ala167Thr, Gly174Asp, and Glu234Gly). The best mutant laccase model showed that the Gly160 and Ala167 are to be found near the water channel; especially the distance of Ala167 to the Cu3a is 14.46 A. This implies that it is likely involved in the formation of water channel and that it helps facilitate the easy incoming and outgoing of water.

  3. Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity

    Science.gov (United States)

    Leonard, Cory A.; Brown, Stacy D.; Hayman, J. Russell

    2013-01-01

    Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA) in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS). Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa, but not Klebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae. Because the genome of A. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system. PMID:23983696

  4. Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity

    Directory of Open Access Journals (Sweden)

    Cory A. Leonard

    2013-01-01

    Full Text Available Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS. Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa, but not Klebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae. Because the genome of A. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system.

  5. 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.

  6. 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.

  7. A mouse model of hereditary coproporphyria identified in an ENU mutagenesis screen

    Directory of Open Access Journals (Sweden)

    Ashlee J. Conway

    2017-08-01

    Full Text Available A genome-wide ethyl-N-nitrosourea (ENU mutagenesis screen in mice was performed to identify novel regulators of erythropoiesis. Here, we describe a mouse line, RBC16, which harbours a dominantly inherited mutation in the Cpox gene, responsible for production of the haem biosynthesis enzyme, coproporphyrinogen III oxidase (CPOX. A premature stop codon in place of a tryptophan at amino acid 373 results in reduced mRNA expression and diminished protein levels, yielding a microcytic red blood cell phenotype in heterozygous mice. Urinary and faecal porphyrins in female RBC16 heterozygotes were significantly elevated compared with that of wild-type littermates, particularly coproporphyrinogen III, whereas males were biochemically normal. Attempts to induce acute porphyric crises were made using fasting and phenobarbital treatment on females. While fasting had no biochemical effect on RBC16 mice, phenobarbital caused significant elevation of faecal coproporphyrinogen III in heterozygous mice. This is the first known investigation of a mutagenesis mouse model with genetic and biochemical parallels to hereditary coproporphyria.

  8. Caffeine enhanced measurement of mutagenesis by low levels of [gamma]-irradiation in human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Puck, T.P.; Johnson, R.; Waldren, C.A. (Eleanor Roosevelt Institute for Cancer Research, Denver, CO (United States)); Morse, H. (Univ. of Colorado Cancer Center, Denver, CO (United States))

    1993-09-01

    The well-known action of caffeine in synergizing mutagenesis (including chromosome aberrations) of agents like ionizing radiation by inhibition of cellular repair processes has been incorporated into a rapid procedure for detection of mutagenicity with high sensitivity. Effects of 5-10 rads of [gamma]-irradiation, which approximate the human lifetime dose accumulation from background radiation, can be detected in a two-day procedure using an immortalized human WBC culture. Chromosomally visible lesions are scored on cells incubated for 2 h after irradiation in the presence and absence of 1.0 mg/ml of caffeine. An eightfold amplification of scorable lesions is achieved over the action of radiation alone. This approach provides a closer approximation to absolute mutagenicity unmitigated by repair processes, which can vary in different situations. It is proposed that mutagenesis testing of this kind, using caffiene or other repair-inhibitory agents, be employed to identify mutagens in their effective concentrations to which human populations may be exposed; to detect agents such as caffeine that may synergize mutagenic actions and pose epidemiologic threats; and to discover effective anti-mutagens. Information derived from the use of such procedures may help prevent cancer and newly acquired genetic disease.

  9. 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.

  10. Lentiviral vector-based insertional mutagenesis identifies genes associated with liver cancer

    Science.gov (United States)

    Ranzani, Marco; Cesana, Daniela; Bartholomae, Cynthia C.; Sanvito, Francesca; Pala, Mauro; Benedicenti, Fabrizio; Gallina, Pierangela; Sergi, Lucia Sergi; Merella, Stefania; Bulfone, Alessandro; Doglioni, Claudio; von Kalle, Christof; Kim, Yoon Jun; Schmidt, Manfred; Tonon, Giovanni; Naldini, Luigi; Montini, Eugenio

    2013-01-01

    Transposons and γ-retroviruses have been efficiently used as insertional mutagens in different tissues to identify molecular culprits of cancer. However, these systems are characterized by recurring integrations that accumulate in tumor cells, hampering the identification of early cancer-driving events amongst bystander and progression-related events. We developed an insertional mutagenesis platform based on lentiviral vectors (LVV) by which we could efficiently induce hepatocellular carcinoma (HCC) in 3 different mouse models. By virtue of LVV’s replication-deficient nature and broad genome-wide integration pattern, LVV-based insertional mutagenesis allowed identification of 4 new liver cancer genes from a limited number of integrations. We validated the oncogenic potential of all the identified genes in vivo, with different levels of penetrance. Our newly identified cancer genes are likely to play a role in human disease, since they are upregulated and/or amplified/deleted in human HCCs and can predict clinical outcome of patients. PMID:23314173

  11. Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.

    Science.gov (United States)

    Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F Peter; Zhang, Huidong

    2016-01-01

    Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, Escherichia coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Laboratory Activity to Promote Student Understanding of UV Mutagenesis and DNA Repair

    Directory of Open Access Journals (Sweden)

    Joshua Ernest Kouassi

    2017-05-01

    Full Text Available Changes in DNA molecules are common, due to the effects of UV light and other external and internal mutagens. Cells have a variety of repair mechanisms which serve to maintain the accuracy of the genetic code. This activity includes a low-cost, safe and technically feasible experiment, which allows students to observe the effects of UV mutagenesis and DNA photorepair in the halophilc archaeon, Haloferax volcanii. An optional extension links this activity to topics of immediate concern to students – how exposure to UVC light contributes to skin cancer risk and the protective effects of sunscreen. Students design and carry out an experiment to test whether SPF 15 sunscreen increases the lethal exposure time for H. volcanii by a factor of 15. Throughout the activity, discussion questions engage students in actively thinking about the biological phenomena and experimental procedures and analysis. This activity is designed for students in college or university genetics, microbiology, or introductory biology courses as well as in high school honors biology courses. Teachers report that this activity was valuable in helping students understand mutagenesis and photorepair and in developing student skills in designing and analyzing experiments.

  13. Role of Base Excision Repair (BER) in Transcription-associated Mutagenesis of Nutritionally Stressed Nongrowing Bacillus subtilis Cell Subpopulations.

    Science.gov (United States)

    Ambriz-Aviña, Verónica; Yasbin, Ronald E; Robleto, Eduardo A; Pedraza-Reyes, Mario

    2016-11-01

    Compelling evidence points to transcriptional processes as important factors contributing to stationary-phase associated mutagenesis. However, it has not been documented whether or not base excision repair mechanisms play a role in modulating mutagenesis under conditions of transcriptional derepression. Here, we report on a flow cytometry-based methodology that employs a fluorescent reporter system to measure at single-cell level, the occurrence of transcription-associated mutations in nutritionally stressed B. subtilis cultures. Using this approach, we demonstrate that (i) high levels of transcription correlates with augmented mutation frequency, and (ii) mutation frequency is enhanced in nongrowing population cells deficient for deaminated (Ung, YwqL) and oxidized guanine (GO) excision repair, strongly suggesting that accumulation of spontaneous DNA lesions enhance transcription-associated mutagenesis.

  14. 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.

  15. 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.

  16. Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge?

    Science.gov (United States)

    Breyer, Didier; Herman, Philippe; Brandenburger, Annick; Gheysen, Godelieve; Remaut, Erik; Soumillion, Patrice; Van Doorsselaere, Jan; Custers, René; Pauwels, Katia; Sneyers, Myriam; Reheul, Dirk

    2009-01-01

    In the European Union, the definition of a GMO is technology-based. This means that a novel organism will be regulated under the GMO regulatory framework only if it has been developed with the use of defined techniques. This approach is now challenged with the emergence of new techniques. In this paper, we describe regulatory and safety issues associated with the use of oligonucleotide-mediated mutagenesis to develop novel organisms. We present scientific arguments for not having organisms developed through this technique fall within the scope of the EU regulation on GMOs. We conclude that any political decision on this issue should be taken on the basis of a broad reflection at EU level, while avoiding discrepancies at international level.

  17. 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.

  18. Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis

    Directory of Open Access Journals (Sweden)

    Chen-Ji Huang

    2011-01-01

    Full Text Available In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123 was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W84, P95, P110, or V129. The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3 were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W84S, P110S and V129L exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 μM−1. Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection.

  19. 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.

  20. 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.

  1. The Glyphosate-Based Herbicide Roundup Does not Elevate Genome-Wide Mutagenesis of Escherichia coli

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    Clayton Tincher

    2017-10-01

    Full Text Available Mutations induced by pollutants may promote pathogen evolution, for example by accelerating mutations conferring antibiotic resistance. Generally, evaluating the genome-wide mutagenic effects of long-term sublethal pollutant exposure at single-nucleotide resolution is extremely difficult. To overcome this technical barrier, we use the mutation accumulation/whole-genome sequencing (MA/WGS method as a mutagenicity test, to quantitatively evaluate genome-wide mutagenesis of Escherichia coli after long-term exposure to a wide gradient of the glyphosate-based herbicide (GBH Roundup Concentrate Plus. The genome-wide mutation rate decreases as GBH concentration increases, suggesting that even long-term GBH exposure does not compromise the genome stability of bacteria.

  2. Directed mutagenesis under the action of mobile elements in unstable lines of Drosophila melanogaster

    Energy Technology Data Exchange (ETDEWEB)

    Gerasimova, T.I.; Mizrokhi, L.Yu.; Obolenkova, L.A.; Georgiev, P.G.

    1985-07-01

    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/sup -2/-10/sup -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/sup 8/ dmB. In situ hybridization with the (/sup 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.

  3. 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

  4. BAX and tumor suppressor TRP53 are important in regulating mutagenesis in spermatogenic cells in mice.

    Science.gov (United States)

    Xu, Guogang; Vogel, Kristine S; McMahan, C Alex; Herbert, Damon C; Walter, Christi A

    2010-12-01

    During the first wave of spermatogenesis, and in response to ionizing radiation, elevated mutant frequencies are reduced to a low level by unidentified mechanisms. Apoptosis is occurring in the same time frame that the mutant frequency declines. We examined the role of apoptosis in regulating mutant frequency during spermatogenesis. Apoptosis and mutant frequencies were determined in spermatogenic cells obtained from Bax-null or Trp53-null mice. The results showed that spermatogenic lineage apoptosis was markedly decreased in Bax-null mice and was accompanied by a significantly increased spontaneous mutant frequency in seminiferous tubule cells compared to that of wild-type mice. Apoptosis profiles in the seminiferous tubules for Trp53-null were similar to control mice. Spontaneous mutant frequencies in pachytene spermatocytes and in round spermatids from Trp53-null mice were not significantly different from those of wild-type mice. However, epididymal spermatozoa from Trp53-null mice displayed a greater spontaneous mutant frequency compared to that from wild-type mice. A greater proportion of spontaneous transversions and a greater proportion of insertions/deletions 15 days after ionizing radiation were observed in Trp53-null mice compared to wild-type mice. Base excision repair activity in mixed germ cell nuclear extracts prepared from Trp53-null mice was significantly lower than that for wild-type controls. These data indicate that BAX-mediated apoptosis plays a significant role in regulating spontaneous mutagenesis in seminiferous tubule cells obtained from neonatal mice, whereas tumor suppressor TRP53 plays a significant role in regulating spontaneous mutagenesis between postmeiotic round spermatid and epididymal spermatozoon stages of spermiogenesis.

  5. Characterization of a new allele of Ames waltzer generated by ENU mutagenesis.

    Science.gov (United States)

    Washington, Jesse L; Pitts, Darrell; Wright, Charles G; Erway, L C; Davis, Rickie R; Alagramam, Kumar

    2005-04-01

    Mutation in the protocadherin 15 (Pcdh15) gene causes hair cell dysfunction and is associated with abnormal stereocilia development. We have characterized the first allele (Pcdh15(av-nmf19)) of Ames waltzer (av) obtained by N-ethyl-N-nitrosourea (ENU) mutagenesis. Pcdh15(av-nmf19) was generated in the Neuroscience Mutagenesis Facility (NMF) at The Jackson Lab (Bar Habor, USA). Pcdh15(av-nmf19) mutants display circling and abnormal swimming behavior along with lack of auditory-evoked brainstem response at the highest intensities tested. Mutation analysis shows base substitution (A--> G) in the consensus splice donor sequence linked to exon 14 resulting in the skipping of exon 14 and the splicing of exon 13-15. This results in the introduction of a stop codon in the coding sequence of exon 15 due to shift in the reading frame. The effect of nmf19 mutation is expected to be severe since the expressed Pcdh15 protein is predicted to truncate in the 5th cadherin domain. Abnormalities of cochlear hair cell stereocilia are apparent in Pcdh15(av-nmf19) mutants near the time of birth and by about P15 (15 days after birth) there is evidence of sensory cell degeneration. Disorganization of outer hair cell stereocilia is observed as early as P2. Inner hair cell stereocilia are also affected, but less severely than those of the outer hair cells. These results are consistent with characteristics of the mutation in the Pcdh15(av-nmf19) allele and they support our previous finding that Protocadherin 15 plays an important role in hair-bundle morphogenesis.

  6. 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.

  7. 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 H2O2 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.

  8. 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)

  9. USP7 Is a Suppressor of PCNA Ubiquitination and Oxidative-Stress-Induced Mutagenesis in Human Cells

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    Shu-ichiro Kashiwaba

    2015-12-01

    Full Text Available Mono-ubiquitinated PCNA activates error-prone DNA polymerases; therefore, strict regulation of PCNA mono-ubiquitination is crucial in avoiding undesired mutagenesis. In this study, we used an in vitro assay system to identify USP7 as a deubiquitinating enzyme of mono-ubiquitinated PCNA. Suppression of USP1, a previously identified PCNA deubiquitinase, or USP7 increased UV- and H2O2-induced PCNA mono-ubiquitination in a distinct and additive manner, suggesting that USP1 and USP7 make different contributions to PCNA deubiquitination in human cells. Cell-cycle-synchronization analyses revealed that USP7 suppression increased H2O2-induced PCNA ubiquitination throughout interphase, whereas USP1 suppression specifically increased ubiquitination in S-phase cells. UV-induced mutagenesis was elevated in USP1-suppressed cells, whereas H2O2-induced mutagenesis was elevated in USP7-suppressed cells. These results suggest that USP1 suppresses UV-induced mutations produced in a manner involving DNA replication, whereas USP7 suppresses H2O2-induced mutagenesis involving cell-cycle-independent processes such as DNA repair.

  10. Insight into the molecular requirements for pathogenicity of Fusarium oxysporum f. sp. lycopersici through large-scale insertional mutagenesis

    NARCIS (Netherlands)

    Michielse, C.B.; van Wijk, R.; Reijnen, L.; Cornelissen, B.J.C.; Rep, M.

    2009-01-01

    Background: Fusarium oxysporum f. sp. lycopersici is the causal agent of vascular wilt disease in tomato. In order to gain more insight into the molecular processes in F. oxysporum necessary for pathogenesis and to uncover the genes involved, we used Agrobacterium-mediated insertional mutagenesis to

  11. Insertional Mutagenesis and Deep Profiling Reveals Gene Hierarchies and a Myc/p53-Dependent Bottleneck in Lymphomagenesis

    NARCIS (Netherlands)

    Huser, C.A.; Gilroy, K.L.; De Ridder, J.; Kilbey, A.; Borland, G.; Mackay, N.; Jenkins, A.; Bell, M.; Herzyk, P.; Van der Weyden, L.

    2014-01-01

    Retroviral insertional mutagenesis (RIM) is a powerful tool for cancer genomics that was combined in this study with deep sequencing (RIM/DS) to facilitate a comprehensive analysis of lymphoma progression. Transgenic mice expressing two potent collaborating oncogenes in the germ line (CD2-MYC,

  12. Characterization of the functional epitope on the urokinase receptor. Complete alanine scanning mutagenesis supplemented by chemical cross-linking

    DEFF Research Database (Denmark)

    Gårdsvoll, Henrik; Gilquin, Bernard; Le Du, Marie Hélène

    2006-01-01

    a comprehensive alanine scanning mutagenesis of uPAR combined with low resolution distance constraints defined within the complex using chemical cross-linkers as molecular rulers. The kinetic rate constants for the interaction between pro-uPA and 244 purified uPAR mutants with single-site replacements were...

  13. 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.

  14. Why eukaryotic cells use introns to enhance gene expression: Splicing reduces transcription-associated mutagenesis by inhibiting topoisomerase I cutting activity

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    Yang Yu-Fei

    2011-05-01

    Full Text Available Abstract Background The costs and benefits of spliceosomal introns in eukaryotes have not been established. One recognized effect of intron splicing is its known enhancement of gene expression. However, the mechanism regulating such splicing-mediated expression enhancement has not been defined. Previous studies have shown that intron splicing is a time-consuming process, indicating that splicing may not reduce the time required for transcription and processing of spliced pre-mRNA molecules; rather, it might facilitate the later rounds of transcription. Because the densities of active RNA polymerase II on most genes are less than one molecule per gene, direct interactions between the splicing apparatus and transcriptional complexes (from the later rounds of transcription are infrequent, and thus unlikely to account for splicing-mediated gene expression enhancement. Presentation of the hypothesis The serine/arginine-rich protein SF2/ASF can inhibit the DNA topoisomerase I activity that removes negative supercoiling of DNA generated by transcription. Consequently, splicing could make genes more receptive to RNA polymerase II during the later rounds of transcription, and thus affect the frequency of gene transcription. Compared with the transcriptional enhancement mediated by strong promoters, intron-containing genes experience a lower frequency of cut-and-paste processes. The cleavage and religation activity of DNA strands by DNA topoisomerase I was recently shown to account for transcription-associated mutagenesis. Therefore, intron-mediated enhancement of gene expression could reduce transcription-associated genome instability. Testing the hypothesis Experimentally test whether transcription-associated mutagenesis is lower in intron-containing genes than in intronless genes. Use bioinformatic analysis to check whether exons flanking lost introns have higher frequencies of short deletions. Implications of the hypothesis The mechanism of intron

  15. 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

  16. 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; Chen, Yijun

    2017-02-15

    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. 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 biosynthetic process, the

  17. 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

    Full Text Available Abstract Background N-ethyl-N-nitrosourea mutagenesis was used to induce a point mutation in C57BL/6 J mice. Pain-related phenotype screening was performed in 915 G3 mice. We report the detection of a heritable recessive mutant in meiotic recombinant N1F1 mice that caused an abnormal pain sensitivity phenotype with spontaneous skin inflammation in the paws and ears. Methods We investigated abnormal sensory processing, neuronal peptides, and behavioral responses after the induction of autoinflammatory disease. Single-nucleotide polymorphism (SNP markers and polymerase chain reaction product sequencing were used to identify the mutation site. Results All affected mice developed paw inflammation at 4–8 weeks. Histological examinations revealed hyperplasia of the epidermis in the inflamed paws and increased macrophage expression in the spleen and paw tissues. Mechanical and thermal nociceptive response thresholds were reduced in the affected mice. Locomotor activity was decreased in affected mice with inflamed hindpaws, and this reduction was attributable to the avoidance of contact of the affected paw with the floor. Motor strength and daily activity in the home cage in the affected mice did not show any significant changes. Although Fos immunoreactivity was normal in the dorsal horn of affected mice, calcitonin gene-related peptide immunoreactivity significantly increased in the deep layer of the dorsal horn. The number of microglia increased in the spinal cord, hippocampus, and cerebral cortex in affected mice, and the proliferation of microglia was maintained for a couple of months. Two hundred eighty-five SNP markers were used to reveal the affected gene locus, which was found on the distal part of chromosome 18. A point mutation was detected at A to G in exon 8 of the pstpip2 gene, resulting in a conserved tyrosine residue at amino acid 180 replaced by cysteine (Y180 C. Conclusions The data provide definitive evidence that a mutation

  18. Predatory Short Selling

    OpenAIRE

    Markus K Brunnermeier; Martin Oehmke

    2013-01-01

    Financial institutions may be vulnerable to predatory short selling. When the stock of a financial institution is shorted aggressively, leverage constraints imposed by short-term creditors can force the institution to liquidate long-term investments at fire sale prices. For financial institutions that are sufficiently close to their leverage constraints, predatory short selling equilibria co-exist with no-liquidation equilibria (the vulnerability region) or may even be the unique equilibrium ...

  19. Over short selling

    NARCIS (Netherlands)

    Peeters, M.

    2012-01-01

    Short selling heeft een ietwat dubieuze reputatie. ‘Aandelen verkopen die je niet hebt’, dat wekt al de suggestie dat er iets niet in de haak is. Verschillende toezichthouders en regelgevers leggen snel verbanden tussen short selling en marktmisbruik. ‘Short is moord’ was zelfs de kop boven een

  20. Natural short sleeper

    Science.gov (United States)

    Sleep - natural short sleeper ... 7 to 9 hours of sleep each night. Short sleepers sleep less than 75% of what is normal for ... T, Lauderdale DS. Trends in the prevalence of short sleepers in the USA: 1975-2006. Sleep. 2010;33(1):37-45. PMID: 20120619 www. ...

  1. Short-circuit logic

    NARCIS (Netherlands)

    Bergstra, J.A.; Ponse, A.

    2010-01-01

    Short-circuit evaluation denotes the semantics of propositional connectives in which the second argument is only evaluated if the first argument does not suffice to determine the value of the expression. In programming, short-circuit evaluation is widely used. A short-circuit logic is a variant of

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

    Science.gov (United States)

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

    2016-01-22

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

  3. Defects in the Error Prevention Oxidized Guanine System Potentiate Stationary-Phase Mutagenesis in Bacillus subtilis▿

    Science.gov (United States)

    Vidales, Luz E.; Cárdenas, Lluvia C.; Robleto, Eduardo; Yasbin, Ronald E.; Pedraza-Reyes, Mario

    2009-01-01

    Previous studies showed that a Bacillus subtilis strain deficient in mismatch repair (MMR; encoded by the mutSL operon) promoted the production of stationary-phase-induced mutations. However, overexpression of the mutSL operon did not completely suppress this process, suggesting that additional DNA repair mechanisms are involved in the generation of stationary-phase-associated mutants in this bacterium. In agreement with this hypothesis, the results presented in this work revealed that starved B. subtilis cells lacking a functional error prevention GO (8-oxo-G) system (composed of YtkD, MutM, and YfhQ) had a dramatic propensity to increase the number of stationary-phase-induced revertants. These results strongly suggest that the occurrence of mutations is exacerbated by reactive oxygen species in nondividing cells of B. subtilis having an inactive GO system. Interestingly, overexpression of the MMR system significantly diminished the accumulation of mutations in cells deficient in the GO repair system during stationary phase. These results suggest that the MMR system plays a general role in correcting base mispairing induced by oxidative stress during stationary phase. Thus, the absence or depression of both the MMR and GO systems contributes to the production of stationary-phase mutants in B. subtilis. In conclusion, our results support the idea that oxidative stress is a mechanism that generates genetic diversity in starved cells of B. subtilis, promoting stationary-phase-induced mutagenesis in this soil microorganism. PMID:19011023

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

    Science.gov (United States)

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

    2012-04-17

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

  5. Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing.

    Science.gov (United States)

    Kowalsky, Caitlin A; Faber, Matthew S; Nath, Aritro; Dann, Hailey E; Kelly, Vince W; Liu, Li; Shanker, Purva; Wagner, Ellen K; Maynard, Jennifer A; Chan, Christina; Whitehead, Timothy A

    2015-10-30

    Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, pertussis toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Apparent resistance to mutagenesis by ionizing radiation, and some other unusual responses

    Energy Technology Data Exchange (ETDEWEB)

    Faberge, A.C.

    1983-11-01

    It is pointed out that in some species it is very difficult or even apparently impossible to get genetic visible marker mutants by means of ionizing radiation. Such results are not as a rule published. Species in which no mutant at all could be obtained, after what seem to be adequate efforts by several geneticists are Limnaea peregra, Rhynchosciara angelae, Coelopa frigida, Oncopeltus fasciatus, Dermestes maculatus. Published data on Sciara, Tribolium, Blattella, in which a relative difficulty in getting mutants is reported, are discussed and compared to many other forms in which obtaining marker mutants is on the contrary easy. What appears to be very abnormal mutagenesis is described in Drosophila busckii, which yielded an unusual frequency of autosomal dominants, and Drosophila nebulosa, in which only very poor mutants could be obtained. Possible reasons for all these departures from the usual are discussed. No firm conclusions are reached, but one is led to consider the possible effects of overall chromosome structure, presence or absence of heterochromatin, and multiplicity at the single locus or single polytene band level. There seems to be no relation between the rates of induction of dominant lethality and of point mutation for visible markers.

  7. CRISPR/Cas9-Mediated In Vitro Mutagenesis in GC-Like B Cells.

    Science.gov (United States)

    Chu, Van Trung; Graf, Robin; Rajewsky, Klaus

    2017-01-01

    The CRISPR/Cas9 technology has developed into a powerful tool for genome editing, both in terms of gene silencing and the insertion of precise mutations. However, the application of CRISPR/Cas9-mediated mutagenesis in primary immune cells, in particular in B cells, is still in its infancy because of the difficulty to deliver the CRISPR/Cas9 system into these cells. Here, we describe a new method to use CRISPR/Cas9 for manipulating genes in germinal center (GC)-like B cells in vitro. We isolated Cas9-expressing B cells from R26-Cas9iGFP/+ mice (expressing Cas9 constitutively from the Rosa26 locus) and mixed them with control B cells. Primary B cells were cultured on CD40L- and BAFF-expressing feeder cells and transduced with retroviral particles expressing the sgRNAs of interest. Using this system, we have achieved complete gene knockouts in up to 92% of activated B cells.

  8. 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.

  9. Enhancement of Biomass and Lipid Productivities of Water Surface-Floating Microalgae by Chemical Mutagenesis

    Directory of Open Access Journals (Sweden)

    Daisuke Nojima

    2017-05-01

    Full Text Available 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.

  10. ENU-induced mutagenesis in grass carp (Ctenopharyngodon idellus by treating mature sperm.

    Directory of Open Access Journals (Sweden)

    Xia-Yun Jiang

    Full Text Available N-ethyl-N-nitrosourea (ENU mutagenesis is a useful approach for genetic improvement of plants, as well as for inducing functional mutants in animal models including mice and zebrafish. In the present study, mature sperm of grass carp (Ctenopharyngodon idellus were treated with a range of ENU concentrations for 45 min, and then wild-type eggs were fertilized. The results indicated that the proportion of embryos with morphological abnormalities at segmentation stage or dead fry at hatching stage increased with increasing ENU dose up to 10 mM. Choosing a dose that was mutagenic, but provided adequate numbers of viable fry, an F1 population was generated from 1 mM ENU-treated sperm for screening purposes. The ENU-treated F1 population showed large variations in growth during the first year. A few bigger mutants with morphologically normal were generated, as compared to the controls. Analysis of DNA from 15 F1 ENU-treated individuals for mutations in partial coding regions of igf-2a, igf-2b, mstn-1, mstn-2, fst-1 and fst-2 loci revealed that most ENU-treated point mutations were GC to AT or AT to GC substitution, which led to nonsense, nonsynonymous and synonymous mutations. The average mutation rate at the examined loci was 0.41%. These results indicate that ENU treatment of mature sperm can efficiently induce point mutations in grass carp, which is a potentially useful approach for genetic improvement of these fish.

  11. In vitro mutagenesis in embryogenic cell suspensions of banana cv. Grande naine (Musa AAA

    Directory of Open Access Journals (Sweden)

    Idalmis Bermúdez-Caraballoso

    2016-04-01

    Full Text Available Somatic embryogenesis is a useful process for clonal propagation and genetic improvement by induction of mutations. This work was carried out with the objective of determining the effect of 60Co source Gamma radiations on embryogenic cell suspensions of banana cv. 'Grande naine' (Musa AAA until conversion to plants. Different doses of radiation (0, 30, 40, 50, 60, 70 and 80 Gy were applied to embryogenic cell suspensions in the multiplication phase and the embryos were later formed, matured and germinated. To determine the ex vitro response of the population of plants obtained these were transferred to greenhouse. The results showed that with somatic embryos formed fresh mass no differences were observed between the effect of the different doses of radiation applied and the control. However, the radiation dose affected the percentage of somatic embryo formation and germination. Plants with phenotypic variations were regenerated with 40 Gy. The results at the greenhouse showed that as radiation doses increased up to 50 Gy, the frequency of variations increased. With higher doses of radiation the survival of the plants was affected.   Keywords: Gamma radiation, in vitro mutagenesis, radiation dose, radiosensibility, somatic embryo

  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. Random mutagenesis of bacterial luciferase: critical role of Glu175 in the control of luminescence decay

    Science.gov (United States)

    2004-01-01

    Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. Luciferases from Vibrio harveyi and Xenorhabdus (Photorhabdus) luminescens have slow decay rates, and those from the Photobacterium genus, such as Photobacterium fisheri, P. phosphoreum and P. leiognathi, have rapid decay rates. By substitution of a 67-amino-acid stretch of P. phosphoreum LuxA in the central region of the LuxA subunit, the ‘slow’ X. luminescens luciferase was converted into a chimaeric luciferase with a significantly more rapid decay rate [Valkova, Szittner and Meighen (1999) Biochemistry 38, 13820–13828]. To understand better the role of specific residues in the classification of luciferases as slow and fast decay, we have conducted random mutagenesis on this region. One of the mutants generated by a single mutation on LuxA at position 175 [E175G (Glu175→Gly)] resulted in the ‘slow decay’ X. luminescens luciferase being converted into a luciferase with a significantly more rapid decay rate. These results indicate the importance of Glu175 in LuxA as a critical residue for differentiating between ‘slow’ and ‘fast’ luciferases and show that this distinction is primarily due to differences in aldehyde affinity and in the decomposition of the luciferase–flavin–oxygen intermediate. PMID:15352872

  14. General mutagenesis of F plasmid TraI reveals its role in conjugative regulation.

    Science.gov (United States)

    Haft, Rembrandt J F; Palacios, Gilberto; Nguyen, Tran; Mally, Manuela; Gachelet, Eliora G; Zechner, Ellen L; Traxler, Beth

    2006-09-01

    Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene traI using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI that can tolerate insertions of a moderate size. We also demonstrate that wild-type TraI, when overexpressed, plays a dominant-negative regulatory role in conjugation, repressing plasmid transfer frequencies approximately 100-fold. Mutant TraI proteins with insertions in a region of approximately 400 residues between the consensus relaxase and helicase sequences did not cause conjugative repression. These unrestrictive TraI variants have normal relaxase activity in vivo, and several have wild-type conjugative functions when expressed at normal levels. We postulate that TraI negatively regulates conjugation by interacting with and sequestering some component of the conjugative apparatus. Our data indicate that the domain responsible for conjugative repression resides in the central region of TraI between the protein's catalytic domains.

  15. Crizotinib-resistant mutants of EML4-ALK identified through an accelerated mutagenesis screen.

    Science.gov (United States)

    Zhang, Sen; Wang, Frank; Keats, Jeffrey; Zhu, Xiaotian; Ning, Yaoyu; Wardwell, Scott D; Moran, Lauren; Mohemmad, Qurish K; Anjum, Rana; Wang, Yihan; Narasimhan, Narayana I; Dalgarno, David; Shakespeare, William C; Miret, Juan J; Clackson, Tim; Rivera, Victor M

    2011-12-01

    Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance. © 2011 John Wiley & Sons A/S.

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

    Science.gov (United States)

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

    2016-01-01

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

  17. Sleeping Beauty insertional mutagenesis in mice identifies drivers of steatosis-associated hepatic tumor.

    Science.gov (United States)

    Tschida, Barbara R; Temiz, Nuri A; Kuka, Timothy P; Lee, Lindsey A; Riordan, Jesse D; Tierrablanca, Carlos A; Hullsiek, Robert; Wagner, Sandra; Hudson, Wendy A; Linden, Michael A; Amin, Khalid; Beckmann, Pauline J; Heuer, Rachel A; Sarver, Aaron L; Yang, Ju Dong; Roberts, Lewis R; Nadeau, Joseph H; Dupuy, Adam J; Keng, Vincent W; Largaespada, David

    2017-10-09

    Hepatic steatosis is a strong risk factor for the development of hepatocellular carcinoma (HCC), yet little is known about the molecular pathology associated with this factor. In this study, we performed a forward genetic screen using Sleeping Beauty (SB) transposon insertional mutagenesis in mice treated to induce hepatic steatosis, and compared the results to human HCC data. In humans, we determined that steatosis increased the proportion of female HCC patients, a pattern also reflected in mice. Our genetic screen identified 203 candidate steatosis-associated HCC genes, many of which are altered in human HCC and are members of established HCC-driving signaling pathways. The protein kinase A/cyclic AMP signaling pathway was altered frequently in mouse and human steatosis-associated HCC. We found that activated PKA expression drove steatosis-specific liver tumorigenesis in a mouse model. Another candidate HCC driver, the N-acetyltransferase NAT10, which we found to be overexpressed in human steatosis-associated HCC and associated with decreased survival in human HCC, also drove liver tumorigenesis in a steatotic mouse model. This study identifies genes and pathways promoting HCC that may represent novel targets for prevention and treatment in the context of hepatic steatosis, an area of rapidly growing clinical significance. Copyright ©2017, American Association for Cancer Research.

  18. 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.

  19. Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis.

    Science.gov (United States)

    Starita, Lea M; Pruneda, Jonathan N; Lo, Russell S; Fowler, Douglas M; Kim, Helen J; Hiatt, Joseph B; Shendure, Jay; Brzovic, Peter S; Fields, Stanley; Klevit, Rachel E

    2013-04-02

    Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify the molecular determinants within E3 ligases that modulate activity, we scored each member of a library of nearly 100,000 protein variants of the murine ubiquitination factor E4B (Ube4b) U-box domain for auto-ubiquitination activity in the presence of the E2 UbcH5c. This assay identified mutations that enhance activity both in vitro and in cellular p53 degradation assays. The activity-enhancing mutations fall into two distinct mechanistic classes: One increases the U-box:E2-binding affinity, and the other allosterically stimulates the formation of catalytically active conformations of the E2∼Ub conjugate. The same mutations enhance E3 activity in the presence of another E2, Ube2w, implying a common allosteric mechanism, and therefore the general applicability of our observations to other E3s. A comparison of the E3 activity with the two different E2s identified an additional variant that exhibits E3:E2 specificity. Our results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity.

  20. Identification of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis.

    Science.gov (United States)

    Fuller, T E; Kennedy, M J; Lowery, D E

    2000-07-01

    P. multocida is the causative agent of several economically significant veterinary diseases occurring in numerous species worldwide. Signature-tagged mutagenesis (STM) is a powerful genetic technique used to simultaneously screen multiple transposon mutants of a pathogen for their inability to survive in vivo. We have designed an STM system based on a mini-Tn10 transposon, chemiluminescent detection and semi-quantitative analysis and have identified transposon insertions into genes of Pasteurella multocida that attenuate virulence in a septicemic mouse model. A bank of 96 transposons containing strongly-hybridizing tags was used to create 19 pools of P. multocida transposon mutants containing approximately 70-90 mutants/pool. A total of 62 mutants were attenuated when checked individually, and 25 unique single transposon insertion mutations were identified from this group. The sequence of the disrupted ORF for each attenuated mutant was determined by either cloning or PCR-amplifying and sequencing the flanking regions. The attenuated mutants contained transposon insertions in genes encoding biosynthetic enzymes, virulence factors, regulatory components and unknown functions. This study should contribute to an understanding of the pathogenic mechanisms by which P. multocida and other pathogens in the Pasteurellaceae family cause disease and identify novel live vaccine candidates and new potential antibiotic targets. Copyright 2000 Academic Press.

  1. An ENU-mutagenesis screen in the mouse: identification of novel developmental gene functions.

    Directory of Open Access Journals (Sweden)

    Carolien Wansleeben

    Full Text Available BACKGROUND: Mutagenesis screens in the mouse have been proven useful for the identification of novel gene functions and generation of interesting mutant alleles. Here we describe a phenotype-based screen for recessive mutations affecting embryonic development. METHODOLOGY/PRINCIPAL FINDINGS: Mice were mutagenized with N-ethyl-N-nitrosourea (ENU and following incrossing the offspring, embryos were analyzed at embryonic day 10.5. Mutant phenotypes that arose in our screen include cardiac and nuchal edema, neural tube defects, situs inversus of the heart, posterior truncation and the absence of limbs and lungs. We isolated amongst others novel mutant alleles for Dll1, Ptprb, Plexin-B2, Fgf10, Wnt3a, Ncx1, Scrib(Scrib, Scribbled homolog [Drosophila] and Sec24b. We found both nonsense alleles leading to severe protein truncations and mutants with single-amino acid substitutions that are informative at a molecular level. Novel findings include an ectopic neural tube in our Dll1 mutant and lung defects in the planar cell polarity mutants for Sec24b and Scrib. CONCLUSIONS/SIGNIFICANCE: Using a forward genetics approach, we have generated a number of novel mutant alleles that are linked to disturbed morphogenesis during development.

  2. 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.

  3. Promoter mutagenesis for fine-tuning expression of essential genes in Mycobacterium tuberculosis.

    Science.gov (United States)

    Boldrin, Francesca; Degiacomi, Giulia; Serafini, Agnese; Kolly, Gaëlle S; Ventura, Marcello; Sala, Claudia; Provvedi, Roberta; Palù, Giorgio; Cole, Stewart T; Manganelli, Riccardo

    2018-01-01

    A range of regulated gene expression systems has been developed for mycobacteria in the last few years to facilitate the study of essential genes, validate novel drug targets and evaluate their vulnerability. Among these, the TetR/Pip-OFF repressible promoter system was successfully used in several mycobacterial species both in vitro and in vivo. In the first version of the system, the repressible promoter was Pptr , a strong Pip-repressible promoter of Streptomyces pristinaespiralis, which might hamper effective downregulation of genes with a low basal expression level. Here, we report an enhanced system that allows more effective control of genes expressed at low level. To this end, we subjected Pptr to targeted mutagenesis and produced 16 different promoters with different strength. Three of them, weaker than the wild-type promoter, were selected and characterized showing that they can indeed improve the performances of TetR/Pip-OFF repressible system both in vitro and in vivo increasing its stringency. Finally, we used these promoters to construct a series of bacterial biosensors with different sensitivity to DprE1 inhibitors and developed a whole-cell screening assay to identify inhibitors of this enzyme. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  4. Insertional Mutagenesis Identifies a STAT3/Arid1b/β-catenin Pathway Driving Neurofibroma Initiation

    Directory of Open Access Journals (Sweden)

    Jianqiang Wu

    2016-03-01

    Full Text Available To identify genes and signaling pathways that initiate Neurofibromatosis type 1 (NF1 neurofibromas, we used unbiased insertional mutagenesis screening, mouse models, and molecular analyses. We mapped an Nf1-Stat3-Arid1b/β-catenin pathway that becomes active in the context of Nf1 loss. Genetic deletion of Stat3 in Schwann cell progenitors (SCPs and Schwann cells (SCs prevents neurofibroma formation, decreasing SCP self-renewal and β-catenin activity. β-catenin expression rescues effects of Stat3 loss in SCPs. Importantly, P-STAT3 and β-catenin expression correlate in human neurofibromas. Mechanistically, P-Stat3 represses Gsk3β and the SWI/SNF gene Arid1b to increase β-catenin. Knockdown of Arid1b or Gsk3β in Stat3fl/fl;Nf1fl/fl;DhhCre SCPs rescues neurofibroma formation after in vivo transplantation. Stat3 represses Arid1b through histone modification in a Brg1-dependent manner, indicating that epigenetic modification plays a role in early tumorigenesis. Our data map a neural tumorigenesis pathway and support testing JAK/STAT and Wnt/β-catenin pathway inhibitors in neurofibroma therapeutic trials.

  5. 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.

  6. Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments.

    Science.gov (United States)

    Kalyoncu, Sibel; Hyun, Jeongmin; Pai, Jennifer C; Johnson, Jennifer L; Entzminger, Kevin; Jain, Avni; Heaner, David P; Morales, Ivan A; Truskett, Thomas M; Maynard, Jennifer A; Lieberman, Raquel L

    2014-09-01

    Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three-dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although noncomplementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts. © 2014 Wiley Periodicals, Inc.

  7. Contribution of increased mutagenesis to the evolution of pollutants-degrading indigenous bacteria.

    Science.gov (United States)

    Ilmjärv, Tanel; Naanuri, Eve; Kivisaar, Maia

    2017-01-01

    Bacteria can rapidly evolve mechanisms allowing them to use toxic environmental pollutants as a carbon source. In the current study we examined whether the survival and evolution of indigenous bacteria with the capacity to degrade organic pollutants could be connected with increased mutation frequency. The presence of constitutive and transient mutators was monitored among 53 pollutants-degrading indigenous bacterial strains. Only two strains expressed a moderate mutator phenotype and six were hypomutators, which implies that constitutively increased mutability has not been prevalent in the evolution of pollutants degrading bacteria. At the same time, a large proportion of the studied indigenous strains exhibited UV-irradiation-induced mutagenesis, indicating that these strains possess error-prone DNA polymerases which could elevate mutation frequency transiently under the conditions of DNA damage. A closer inspection of two Pseudomonas fluorescens strains PC20 and PC24 revealed that they harbour genes for ImuC (DnaE2) and more than one copy of genes for Pol V. Our results also revealed that availability of other nutrients in addition to aromatic pollutants in the growth environment of bacteria affects mutagenic effects of aromatic compounds. These results also implied that mutagenicity might be affected by a factor of how long bacteria have evolved to use a particular pollutant as a carbon source.

  8. Contribution of increased mutagenesis to the evolution of pollutants-degrading indigenous bacteria.

    Directory of Open Access Journals (Sweden)

    Tanel Ilmjärv

    Full Text Available Bacteria can rapidly evolve mechanisms allowing them to use toxic environmental pollutants as a carbon source. In the current study we examined whether the survival and evolution of indigenous bacteria with the capacity to degrade organic pollutants could be connected with increased mutation frequency. The presence of constitutive and transient mutators was monitored among 53 pollutants-degrading indigenous bacterial strains. Only two strains expressed a moderate mutator phenotype and six were hypomutators, which implies that constitutively increased mutability has not been prevalent in the evolution of pollutants degrading bacteria. At the same time, a large proportion of the studied indigenous strains exhibited UV-irradiation-induced mutagenesis, indicating that these strains possess error-prone DNA polymerases which could elevate mutation frequency transiently under the conditions of DNA damage. A closer inspection of two Pseudomonas fluorescens strains PC20 and PC24 revealed that they harbour genes for ImuC (DnaE2 and more than one copy of genes for Pol V. Our results also revealed that availability of other nutrients in addition to aromatic pollutants in the growth environment of bacteria affects mutagenic effects of aromatic compounds. These results also implied that mutagenicity might be affected by a factor of how long bacteria have evolved to use a particular pollutant as a carbon source.

  9. 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...

  10. FHIT loss-induced DNA damage creates optimal APOBEC substrates: Insights into APOBEC-mediated mutagenesis.

    Science.gov (United States)

    Waters, Catherine E; Saldivar, Joshua C; Amin, Zaynab A; Schrock, Morgan S; Huebner, Kay

    2015-02-20

    APOBEC cytidine deaminase activity is a major source of hypermutation in cancer. But previous studies have shown that the TC context signature of these enzymes is not observed in sizable fractions of cancers with overexpression of APOBEC, suggesting that cooperating factors that contribute to this mutagenesis should be identified. The fragile histidine triad protein (Fhit) is a tumor suppressor and DNA caretaker that is deleted or silenced in >50% of cancers. Loss of Fhit protein activity causes replication stress through reduced Thymidine Kinase 1 expression, increased DNA breaks, and global genome instability in normal and cancer cells. Using data from The Cancer Genome Atlas (TCGA), we show that FHIT-low/APOBEC3B-high expressing lung adenocarcinomas display significantly increased numbers of APOBEC signature mutations. Tumor samples in this cohort with normal FHIT expression do not exhibit APOBEC hypermutation, despite having high APOBEC3B expression. In vitro, silencing Fhit expression elevates APOBEC3B-directed C > T mutations in the TP53 gene. Furthermore, inhibition of Fhit loss-induced DNA damage via thymidine supplementation decreases the TP53 mutation burden in FHIT-low/APOBEC3B-high cells. We conclude that APOBEC3B overexpression and Fhit-loss induced DNA damage are independent events that, when occurring together, result in a significantly increased frequency of APOBEC-induced mutations that drive cancer progression.

  11. Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli.

    Directory of Open Access Journals (Sweden)

    Régine Janel-Bintz

    2017-07-01

    Full Text Available It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM, exhibits several characteristics distinct from mutations that occur within the course of replication: i following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min than replication dependent mutations (t ½ ≈ 80-100 min ii NERiM specifically requires DNA Pol IV in addition to Pol V iii NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication.

  12. 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.

  13. Signature-Tagged Mutagenesis of Pasteurella multocida Identifies Mutants Displaying Differential Virulence Characteristics in Mice and Chickens

    OpenAIRE

    Harper, Marina; Boyce, John D.; Wilkie, Ian W.; Adler, Ben

    2003-01-01

    Pasteurella multocida is the causative agent of fowl cholera in birds. Signature-tagged mutagenesis (STM) was used to identify potential virulence factors in a mouse septicemia disease model and a chicken fowl cholera model. A library of P. multocida mutants was constructed with a modified Tn916 and screened for attenuation in both animal models. Mutants identified by the STM screening were confirmed as attenuated by competitive growth assays in both chickens and mice. Of the 15 mutants ident...

  14. Systematic identification of genetic loci required for polymyxin resistance in Campylobacter jejuni using an efficient in vivo transposon mutagenesis system.

    Science.gov (United States)

    Lin, Jun; Wang, Ying; Hoang, Ky Van

    2009-03-01

    The aim of this study was to identify genetic loci required for polymyxin (PM) resistance in Campylobacter jejuni using an efficient in vivo random mutagenesis system. PM has been widely used as a model peptide to examine mechanisms of bacterial resistance to antimicrobial peptides (AMPs), the major effectors of host innate immunity and also candidates for a new generation of antibiotics. In this study, a commercially available transposon mutagenesis approach (EZ-Tn5 Transposome; Epicentre, Madison, WI) was evaluated and used to systematically identify Campylobacter mutants with increased susceptibility to PM. This simple, yet efficient, transposon mutagenesis approach identified 12 mutants representing seven different genes of C. jejuni 81-176 involved in acquired PM resistance. Backcrossing of the transposon mutations into the parent strain confirmed that the PM-sensitive phenotype in each mutant was linked to the gene with a specific transposon insertion. The genes are identified as being involved in the synthesis of cell-surface carbohydrates, modification of intracellular targets, signal transduction, and modulation of transmembrane potential. The mutant with the highest susceptibility to PM contains a transposon insertion in a putative galU gene that is essential for production of uridine diphosphate glucose (UDP)-glucose, a precursor required for lipooligosaccharide (LOS) synthesis. LOS analysis by tricine SDSPAGE showed significant truncation of the LOS core structure in the galU mutant. Susceptibility assays also indicated that GalU contributed C. jejuni resistance to some natural AMPs. Complementation of the galU mutant in trans fully restored LOS synthesis and resistance to the levels of the parent strain. Together, these results define seven C. jejuni genetic loci that will be useful for characterizing the molecular basis of Campylobacter resistance to PM and natural AMPs, and also highlight the usefulness of the in vivo mutagenesis approach for

  15. The role of Dbf4-dependent protein kinase in DNA polymerase ζ-dependent mutagenesis in Saccharomyces cerevisiae.

    Science.gov (United States)

    Brandão, Luis N; Ferguson, Rebecca; Santoro, Irma; Jinks-Robertson, Sue; Sclafani, Robert A

    2014-08-01

    The yeast Dbf4-dependent kinase (DDK) (composed of Dbf4 and Cdc7 subunits) is an essential, conserved Ser/Thr protein kinase that regulates multiple processes in the cell, including DNA replication, recombination and induced mutagenesis. Only DDK substrates important for replication and recombination have been identified. Consequently, the mechanism by which DDK regulates mutagenesis is unknown. The yeast mcm5-bob1 mutation that bypasses DDK's essential role in DNA replication was used here to examine whether loss of DDK affects spontaneous as well as induced mutagenesis. Using the sensitive lys2ΔA746 frameshift reversion assay, we show DDK is required to generate "complex" spontaneous mutations, which are a hallmark of the Polζ translesion synthesis DNA polymerase. DDK co-immunoprecipitated with the Rev7 regulatory, but not with the Rev3 polymerase subunit of Polζ. Conversely, Rev7 bound mainly to the Cdc7 kinase subunit and not to Dbf4. The Rev7 subunit of Polζ may be regulated by DDK phosphorylation as immunoprecipitates of yeast Cdc7 and also recombinant Xenopus DDK phosphorylated GST-Rev7 in vitro. In addition to promoting Polζ-dependent mutagenesis, DDK was also important for generating Polζ-independent large deletions that revert the lys2ΔA746 allele. The decrease in large deletions observed in the absence of DDK likely results from an increase in the rate of replication fork restart after an encounter with spontaneous DNA damage. Finally, nonepistatic, additive/synergistic UV sensitivity was observed in cdc7Δ pol32Δ and cdc7Δ pol30-K127R,K164R double mutants, suggesting that DDK may regulate Rev7 protein during postreplication "gap filling" rather than during "polymerase switching" by ubiquitinated and sumoylated modified Pol30 (PCNA) and Pol32. Copyright © 2014 by the Genetics Society of America.

  16. Differences in temporal aspects of mutagenesis and cytotoxicity in Chinese hamster cells treated with methylating agents and thymidine.

    Science.gov (United States)

    Peterson, A R; Peterson, H

    1982-01-01

    Equitoxic concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MeMes) produced different frequencies of 8-azaguanine-resistant mutants and different amounts of N7-methylguanine, O6-methylguanine (m6G), and N3-methyladenine in the DNA of V79 Chinese hamster cells. Thus, neither the cytotoxicities nor the mutagenicities of these methylating agents could be attributed solely to nitrogen or to oxygen methylations in the DNA. However, MNNG produced 12-fold more m6G and 5-fold more mutants than did MeMes, indicating that a substantial part of the MNNG-induced mutations resulted from m6G--thymine mispairing during DNA replication. The expression as mutants of mutagenic oxygen methylations in the DNA of cells treated with MNNG was enhanced by thymidine (dThd) and deoxycytidine (dCyd), but these nucleosides did not significantly enhance MeMes-induced mutagenesis. The cytotoxicities of MNNG and MeMes were also increased by 10 microM dThd in proportion to the amount of m6G in the DNA. These increases in cytotoxicity were abolished by dCyd, which did not greatly reduce the dThd-induced enhancements of mutagenesis. Moreover, when dThd was present only during the 2-hr treatment with MNNG, maximal cytotoxicity occurred, but MNNG-induced mutagenesis was not increased. Maximal mutagenesis occurred when the dThd was present throughout the first doubling time of the MNNG-treated cells. Thus, the expression of the cytotoxicity and the mutagenicity associated with m6G in the DNA of V79 cells occurred by quite different mechanisms. PMID:6951203

  17. Sleeping Beauty Transposon Mutagenesis as a Tool for Gene Discovery in the NOD Mouse Model of Type 1 Diabetes.

    Science.gov (United States)

    Elso, Colleen M; Chu, Edward P F; Alsayb, May A; Mackin, Leanne; Ivory, Sean T; Ashton, Michelle P; Bröer, Stefan; Silveira, Pablo A; Brodnicki, Thomas C

    2015-10-04

    A number of different strategies have been used to identify genes for which genetic variation contributes to type 1 diabetes (T1D) pathogenesis. Genetic studies in humans have identified >40 loci that affect the risk for developing T1D, but the underlying causative alleles are often difficult to pinpoint or have subtle biological effects. A complementary strategy to identifying "natural" alleles in the human population is to engineer "artificial" alleles within inbred mouse strains and determine their effect on T1D incidence. We describe the use of the Sleeping Beauty (SB) transposon mutagenesis system in the nonobese diabetic (NOD) mouse strain, which harbors a genetic background predisposed to developing T1D. Mutagenesis in this system is random, but a green fluorescent protein (GFP)-polyA gene trap within the SB transposon enables early detection of mice harboring transposon-disrupted genes. The SB transposon also acts as a molecular tag to, without additional breeding, efficiently identify mutated genes and prioritize mutant mice for further characterization. We show here that the SB transposon is functional in NOD mice and can produce a null allele in a novel candidate gene that increases diabetes incidence. We propose that SB transposon mutagenesis could be used as a complementary strategy to traditional methods to help identify genes that, when disrupted, affect T1D pathogenesis. Copyright © 2015 Elso et al.

  18. Sleeping Beauty Transposon Mutagenesis as a Tool for Gene Discovery in the NOD Mouse Model of Type 1 Diabetes

    Science.gov (United States)

    Elso, Colleen M.; Chu, Edward P. F.; Alsayb, May A.; Mackin, Leanne; Ivory, Sean T.; Ashton, Michelle P.; Bröer, Stefan; Silveira, Pablo A.; Brodnicki, Thomas C.

    2015-01-01

    A number of different strategies have been used to identify genes for which genetic variation contributes to type 1 diabetes (T1D) pathogenesis. Genetic studies in humans have identified >40 loci that affect the risk for developing T1D, but the underlying causative alleles are often difficult to pinpoint or have subtle biological effects. A complementary strategy to identifying “natural” alleles in the human population is to engineer “artificial” alleles within inbred mouse strains and determine their effect on T1D incidence. We describe the use of the Sleeping Beauty (SB) transposon mutagenesis system in the nonobese diabetic (NOD) mouse strain, which harbors a genetic background predisposed to developing T1D. Mutagenesis in this system is random, but a green fluorescent protein (GFP)-polyA gene trap within the SB transposon enables early detection of mice harboring transposon-disrupted genes. The SB transposon also acts as a molecular tag to, without additional breeding, efficiently identify mutated genes and prioritize mutant mice for further characterization. We show here that the SB transposon is functional in NOD mice and can produce a null allele in a novel candidate gene that increases diabetes incidence. We propose that SB transposon mutagenesis could be used as a complementary strategy to traditional methods to help identify genes that, when disrupted, affect T1D pathogenesis. PMID:26438296

  19. Genome-wide maps of alkylation damage, repair, and mutagenesis in yeast reveal mechanisms of mutational heterogeneity.

    Science.gov (United States)

    Mao, Peng; Brown, Alexander J; Malc, Ewa P; Mieczkowski, Piotr A; Smerdon, Michael J; Roberts, Steven A; Wyrick, John J

    2017-10-01

    DNA base damage is an important contributor to genome instability, but how the formation and repair of these lesions is affected by the genomic landscape and contributes to mutagenesis is unknown. Here, we describe genome-wide maps of DNA base damage, repair, and mutagenesis at single nucleotide resolution in yeast treated with the alkylating agent methyl methanesulfonate (MMS). Analysis of these maps revealed that base excision repair (BER) of alkylation damage is significantly modulated by chromatin, with faster repair in nucleosome-depleted regions, and slower repair and higher mutation density within strongly positioned nucleosomes. Both the translational and rotational settings of lesions within nucleosomes significantly influence BER efficiency; moreover, this effect is asymmetric relative to the nucleosome dyad axis and is regulated by histone modifications. Our data also indicate that MMS-induced mutations at adenine nucleotides are significantly enriched on the nontranscribed strand (NTS) of yeast genes, particularly in BER-deficient strains, due to higher damage formation on the NTS and transcription-coupled repair of the transcribed strand (TS). These findings reveal the influence of chromatin on repair and mutagenesis of base lesions on a genome-wide scale and suggest a novel mechanism for transcription-associated mutation asymmetry, which is frequently observed in human cancers. © 2017 Mao et al.; Published by Cold Spring Harbor Laboratory Press.

  20. A new Penicillium echinulatum strain with faster cellulase secretion obtained using hydrogen peroxide mutagenesis and screening with 2‐deoxyglucose

    National Research Council Canada - National Science Library

    Dillon, A.J.P; Bettio, M; Pozzan, F.G; Andrighetti, T; Camassola, M

    2011-01-01

    Aims:  The aim of this study is to improve cellulase production and secretion by Penicillium echinulatum using mutagenesis and selection in association with microfermentation and microanalysis methods...

  1. 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.

  2. A high-throughput colorimetric assay for screening halohydrin dehalogenase saturation mutagenesis libraries.

    Science.gov (United States)

    Tang, Lixia; Li, Yang; Wang, Xiong

    2010-06-01

    Here we have reported a high throughput pH indicator-based assay to measure the activity of halohydrin dehalogenases (HheC). The assay relies upon the absorbance change at 560nm and the visual color change of phenol red in a weakly buffered system, due to the release of protons from the enzyme-catalyzed ring-closure reactions. The assay can be performed in a microplate format using whole cells, making the assay simple and robust. Thus, it is suitable for library screening. The assay has been further validated using two previously studied HheC variants, D80N and W249F, which exhibit 200-fold lower and 2-fold higher k(cat) values, respectively, toward 1,3-dichloro-2-propanol than the wild-type HheC. In addition, a saturation mutagenesis library of HheC was screened using the developed assay for its ability to efficiently catalyze the conversion of 1,3-dichloro-2-propanol. After screening of 500 colonies, one mutant W139C was identified and was further purified and characterized. Kinetic analysis indicates that the resulting mutant shows 2- and 5-fold improvement in k(cat) value toward 1,3-DCP and (R,S)-p-nitro-2-bromo-1-phenylethanol, respectively, although it exhibits higher K(m) values than the wild-type enzyme. The method described herein represents a useful tool given the need for the high throughput screening of halohydrin dehalogenase mutants. 2010 Elsevier B.V. All rights reserved.

  3. Mapping the HLA-DO/HLA-DM complex by FRET and mutagenesis.

    Science.gov (United States)

    Yoon, Taejin; Macmillan, Henriette; Mortimer, Sarah E; Jiang, Wei; Rinderknecht, Cornelia H; Stern, Lawrence J; Mellins, Elizabeth D

    2012-07-10

    HLA-DO (DO) is a nonclassic class II heterodimer that inhibits the action of the class II peptide exchange catalyst, HLA-DM (DM), and influences DM localization within late endosomes and exosomes. In addition, DM acts as a chaperone for DO and is required for its egress from the endoplasmic reticulum (ER). These reciprocal functions are based on direct DO/DM binding, but the topology of DO/DM complexes is not known, in part, because of technical limitations stemming from DO instability. We generated two variants of recombinant soluble DO with increased stability [zippered DOαP11A (szDOv) and chimeric sDO-Fc] and confirmed their conformational integrity and ability to inhibit DM. Notably, we found that our constructs, as well as wild-type sDO, are inhibitory in the full pH range where DM is active (4.7 to ∼6.0). To probe the nature of DO/DM complexes, we used intermolecular fluorescence resonance energy transfer (FRET) and mutagenesis and identified a lateral surface spanning the α1 and α2 domains of szDO as the apparent binding site for sDM. We also analyzed several sDM mutants for binding to szDOv and susceptibility to DO inhibition. Results of these assays identified a region of DM important for interaction with DO. Collectively, our data define a putative binding surface and an overall orientation of the szDOv/sDM complex and have implications for the mechanism of DO inhibition of DM.

  4. Gene-trap mutagenesis identifies mammalian genes contributing to intoxication by Clostridium perfringens ε-toxin.

    Directory of Open Access Journals (Sweden)

    Susan E Ivie

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

  5. 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.

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

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    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.

  7. Pre-breeding of lentil (Lens culinaris Medik.) for herbicide resistance through seed mutagenesis.

    Science.gov (United States)

    Rizwan, Muhammad; Aslam, Muhammad; Asghar, Muhammad Jawad; Abbas, Ghulam; Shah, Tariq Mahmud; Shimelis, Hussein

    2017-01-01

    Lentil is a poor competitor of weeds and its sensitivity to herbicides is a major hurdle for large scale production. The present study was conducted to select herbicide resistant lentil genotypes through seed mutagenesis. Seeds of three advanced lentil genotypes (LPP 11001, LPP 11100 and LPP 11116) were treated with two different concentrations of ethyl methanesulfonate (EMS; 0.1 and 0.2%), hydrazine hydrate (HH; 0.02 and 0.03%) and sodium azide (SA; 0.01 and 0.02%) to develop M1 seed. The M2 was screened against two herbicides including Ally Max 28.6% SG (X = 34.58 g/ha and 1.5X = 51.87 g/ha) and Atlantis 3.6% WG (X = 395.2 g/ha and 1.5X = 592.8 g/ha) using the following three screening methods: post plant emergence (PPE), pre-plant incorporation (PPI) and seed priming (SP). Data were recorded on survival index and survival percentage from each experimental unit of every population. Plants in all populations were categorized following their reaction to herbicides. The newly developed populations showed greater variation for herbicide resistance when compared to their progenitors. Phenotypic traits were significantly reduced in all the screening environments. Overall, 671 herbicide resistant mutants were selected from all testing environments. The seeds from selected plants were re-mutagenized at 150 Gy of gamma radiation and evaluated against higher dose of herbicides. This allowed selection of 134 herbicide resistant mutants. The selected mutants are useful germplasm for herbicide resistance breeding of lentil.

  8. 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.

  9. Transposon mutagenesis identifies genes and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma

    Science.gov (United States)

    Kodama, Takahiro; Newberg, Justin Y.; Kodama, Michiko; Rangel, Roberto; Yoshihara, Kosuke; Tien, Jean C.; Parsons, Pamela H.; Wu, Hao; Finegold, Milton J.; Copeland, Neal G.; Jenkins, Nancy A.

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets. PMID:27247392

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

    Science.gov (United States)

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

    2013-01-01

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

  11. 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.

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

    Science.gov (United States)

    Rasmussen, Tim; Rasmussen, Akiko; Singh, Shivani; Galbiati, Heloisa; Edwards, Michelle D; Miller, Samantha; Booth, Ian R

    2015-07-28

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

  13. Effect of mutagenesis on the stereochemistry of enoyl-CoA hydratase.

    Science.gov (United States)

    Feng, Yuguo; Hofstein, Hilary A; Zwahlen, Jacque; Tonge, Peter J

    2002-10-22

    Enoyl-CoA hydratase catalyzes the hydration of trans-2-crotonyl-CoA to 3(S)-HB-CoA, 3(S)-hydroxybutyryl-CoA with a stereospecificity (k(S)/k(R)) of 400000 to 1 [Wu, W. J., Feng, Y., He, X., Hofstein, H. S., Raleigh, D. P., and Tonge, P. J. (2000) J. Am. Chem. Soc. 122, 3987-3994]. Replacement of E164, one of the catalytic glutamates in the active site, with either aspartate or glutamine reduces the rate of formation of the 3(S) product enantiomer (k(S)) without affecting the rate of formation of the 3(R) product (k(R)). Consequently, k(S)/k(R) is 1000 and 0.33 for E164D and E164Q, respectively. In contrast, mutagenesis of E144, the second catalytic glutamate, reduces the rate of formation of both product enantiomers. Thus, only E144 is required for the formation of 3(R)-HB-CoA, 3(R)-hydroxybutyryl-CoA. Modeling studies together with analysis of alpha-proton exchange rates and experiments with crotonyl-oxyCoA, a substrate analogue in which the alpha-proton acidity has been reduced 10000-fold, support a mechanism of 3(R)-hydroxybutyryl-CoA formation that involves the E144-catalyzed stepwise addition of water to crotonyl-CoA which is bound in an s-trans conformation in the active site. Finally, we also demonstrate that hydrogen bonds in the oxyanion hole, provided by the backbone amide groups of G141 and A98, are important for the formation of both product enantiomers.

  14. Cytadherence-deficient mutants of Mycoplasma gallisepticum generated by transposon mutagenesis.

    Science.gov (United States)

    Mudahi-Orenstein, Sigalit; Levisohn, Sharon; Geary, Steven J; Yogev, David

    2003-07-01

    Cytadherence-related molecules of Mycoplasma gallisepticum strain R-low were identified by Tn4001 transposon mutagenesis with the hemadsorption (HA) assay as an indicator for cytadherence. Three Gm(r) HA-negative (HA(-)) colonies displaying a stable HA(-) phenotype through several successive generations in which gentamicin selection was maintained were isolated from four independent transformation experiments and characterized. Southern blot analysis showed that the transposon was inserted as a single copy within the genome of each of the HA(-) mutants, suggesting that the transposon insertion was directly responsible for their inability to attach to erythrocytes. Sequence analysis of the transposon insertion sites revealed that in two mutants, the transposon was inserted at two distinct sites within the gapA structural gene. In the third mutant, the insertion was mapped within the crmA gene, which is located immediately downstream of the gapA gene as part of the same operon. In vitro attachment experiments with the MRC-5 human lung fibroblast cell line showed that the cytadherence capabilities of the HA(-) mutants were less than 25% those of original strain R. Experimental infection of chickens, the natural host of M. gallisepticum, with each of the three mutants demonstrated significantly impaired colonization and host responses. These data demonstrate conclusively the role of both GapA and CrmA proteins in the adherence of M. gallisepticum to host cells in model systems and in vivo colonization. Furthermore, these results underscore the relevance of in vitro cytadherence model systems for studying the pathogenesis of natural infections in chickens.

  15. Random mutagenesis reveals residues of JAK2 critical in evading inhibition by a tyrosine kinase inhibitor.

    Directory of Open Access Journals (Sweden)

    Michael R Marit

    Full Text Available The non-receptor tyrosine kinase JAK2 is implicated in a group of myeloproliferative neoplasms including polycythemia vera, essential thrombocythemia, and primary myelofibrosis. JAK2-selective inhibitors are currently being evaluated in clinical trials. Data from drug-resistant chronic myeloid leukemia patients demonstrate that treatment with a small-molecule inhibitor generates resistance via mutation or amplification of BCR-ABL. We hypothesize that treatment with small molecule inhibitors of JAK2 will similarly generate inhibitor-resistant mutants in JAK2.In order to identify inhibitor-resistant JAK2 mutations a priori, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signaling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor.Mutations were found exclusively in the kinase domain of JAK2. The panel of mutations conferred resistance to high concentrations of inhibitor accompanied by sustained activation of the Stat5, Erk1/2, and Akt pathways. Using a JAK2 substrate, enhanced catalytic activity of the mutant JAK2 kinase was observed in inhibitor concentrations 200-fold higher than is inhibitory to the wild-type protein. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor, validating the use of TEL-JAK2 in the initial screen. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles, and the design of next-generation JAK2 inhibitors should consider the location of mutations arising in inhibitor-resistant screens.

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

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    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. Gene transfer and genome-wide insertional mutagenesis by retroviral transduction in fish stem cells.

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    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.

  18. Agrobacterium-mediated transformation of Guignardia citricarpa: an efficient tool to gene transfer and random mutagenesis.

    Science.gov (United States)

    Rodrigues, Maria Beatriz Calderan; Fávaro, Léia Cecília de Lima; Pallu, Ana Paula de Souza; Ferreira, Anderson; Sebastianes, Fernanda de Souza; Rodrigues, Maria Juliana Calderan; Spósito, Marcel Bellato; de Araújo, Welington Luiz; Pizzirani-Kleiner, Aline Aparecida

    2013-01-01

    Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone - AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 μM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant-pathogen interaction. Copyright © 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  19. 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.

  20. 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

  1. ENU Mutagenesis Reveals a Novel Phenotype of Reduced Limb Strength in Mice Lacking Fibrillin 2

    Science.gov (United States)

    Miller, Gaynor; Neilan, Monica; Chia, Ruth; Gheryani, Nabeia; Holt, Natalie; Charbit, Annabelle; Wells, Sara; Tucci, Valter; Lalanne, Zuzanne; Denny, Paul; Fisher, Elizabeth M. C.; Cheeseman, Michael; Askew, Graham N.; Dear, T. Neil

    2010-01-01

    Background 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. Methodology/Principal Findings 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, Fbn2fp, 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. Conclusions 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. PMID

  2. 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.

  3. Gene transfer and genome-wide insertional mutagenesis by retroviral transduction in fish stem cells.

    Science.gov (United States)

    Liu, Qizhi; Wang, Yunzhi; Lin, Fan; Zhang, Lei; Li, Yan; Ge, Ruowen; Hong, Yunhan

    2015-01-01

    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.

  4. 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.

  5. Tryptophan Scanning Mutagenesis Identifies the Molecular Determinants of Distinct Barttin Functions*

    Science.gov (United States)

    Wojciechowski, Daniel; Fischer, Martin; Fahlke, Christoph

    2015-01-01

    CLC-K chloride channels are expressed in the kidney and in the inner ear and require the accessory subunit barttin for proper function and membrane insertion. Barttin exerts multiple functions on CLC-proteins: it modifies protein stability and intracellular trafficking as well as channel activity, ion conduction, and gating. So far, the molecular determinants of these distinct barttin functions have remained elusive. Here we performed serial perturbation mutagenesis to identify the sequence determinants of barttin function. Barttin consists of two transmembrane helices followed by a long intracellular carboxyl terminus, and earlier work demonstrated that the transmembrane core of barttin suffices for most effects on the α-subunit. We individually substituted every amino acid of the predicted transmembrane core (amino acids 9–26 and 35–55) with tryptophan, co-expressed mutant barttin with hClC-Ka or V166E rClC-K1, and characterized CLC-K/barttin channels by patch clamp techniques, biochemistry, and confocal microscopy. The majority of mutations left the chaperone function of barttin, i.e. the effects on endoplasmic reticulum exit and surface membrane insertion, unaffected. In contrast, tryptophan insertion at multiple positions resulted in impaired activity of hClC-Ka/barttin and changes in gating of V166E rClC-K1/barttin. These results demonstrate that mutations in a cluster of hydrophobic residues within transmembrane domain 1 affect barttin-CLC-K interaction and impair gating modification by the accessory subunit. Whereas tight interaction is necessary for functional modification, even impaired association of barttin and CLC-K suffices for normal intracellular trafficking. Our findings allow definition of a likely interaction surface and clarify the mechanisms underlying CLC-K channel modification by barttin. PMID:26063802

  6. Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs.

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    Finola E Moore

    Full Text Available Zinc Finger Nucleases (ZFNs made by Context-Dependent Assembly (CoDA and Transcription Activator-Like Effector Nucleases (TALENs provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%-76.8% compared to 1.1%-3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

  7. Induction of apomixis and fixation of heterosis in Egyptian rice Hybrid1 line using colchicine mutagenesis

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    Reda M. Gaafar

    2017-06-01

    Full Text Available It is known that hybrid rice yields 15–20% over inbred varieties in first generation because of heterosis. However, heterosis is normally broken due to segregation. Applying apomixis produces plants as a clone of mother plant and overcomes the problem of breaking heterosis. In order to fix heterosis in the Egyptian rice Hybrid1, their seeds were mutagenized in 0.2% colchicine for two time periods 24 and 50 h. After colchicine mutagenesis, rice seedlings were grown in the field till maturation and the resulted M1 seeds were sown in season 2 and plants were selected based on yield and homogeneity. Then, seeds were sown to be evaluated in season 3. Pollen fertility test, esterase isozyme analysis, and flow cytometry seed screening were performed to confirm the results of field selection of populations identical to control. Pollen fertility examination was performed on the populations of the third season. Pollens of populations 304, 298, 292, 284, 281, 154 and 149 were found to be completely sterile. However, these plants had high seed set percentage. The flow cytometry screening of the six yield-based identical populations and the control seeds showed that populations 220, 339, 351 and 298 have higher nuclear DNA content (C2 than untreated hybrid (C2 & C3. Results of flow cytometry clearly showed that population 298 has one peak (C2 and its endosperm was formed autonomously without fertilization. Although its pollen grains were sterile, it showed high seed set percentage. This indicates that heterosis was completely fixed by apomixis in this population.

  8. The HIV mutation browser: a resource for human immunodeficiency virus mutagenesis and polymorphism data.

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    Norman E Davey

    2014-12-01

    Full Text Available Huge research effort has been invested over many years to determine the phenotypes of natural or artificial mutations in HIV proteins--interpretation of mutation phenotypes is an invaluable source of new knowledge. The results of this research effort are recorded in the scientific literature, but it is difficult for virologists to rapidly find it. Manually locating data on phenotypic variation within the approximately 270,000 available HIV-related research articles, or the further 1,500 articles that are published each month is a daunting task. Accordingly, the HIV research community would benefit from a resource cataloguing the available HIV mutation literature. We have applied computational text-mining techniques to parse and map mutagenesis and polymorphism information from the HIV literature, have enriched the data with ancillary information and have developed a public, web-based interface through which it can be intuitively explored: the HIV mutation browser. The current release of the HIV mutation browser describes the phenotypes of 7,608 unique mutations at 2,520 sites in the HIV proteome, resulting from the analysis of 120,899 papers. The mutation information for each protein is organised in a residue-centric manner and each residue is linked to the relevant experimental literature. The importance of HIV as a global health burden advocates extensive effort to maximise the efficiency of HIV research. The HIV mutation browser provides a valuable new resource for the research community. The HIV mutation browser is available at: http://hivmut.org.

  9. 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.

  10. 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

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    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.

  11. The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines

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    Tatjana P Kristensen

    2014-03-01

    Full Text Available The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural

  12. Mutagenesis of human immunodeficiency virus reverse transcriptase p51 subunit defines residues contributing to vinylogous urea inhibition of ribonuclease H activity.

    Science.gov (United States)

    Chung, Suhman; Miller, Jennifer T; Johnson, Barry C; Hughes, Stephen H; Le Grice, Stuart F J

    2012-02-03

    The vinylogous urea, NSC727447, was proposed to allosterically inhibit ribonuclease H (RNase H) activity of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) by interacting with the thumb subdomain of its non-catalytic p51 subunit. Proximity of the p51 thumb to the p66 RNase H domain implied that inhibitor binding altered active site geometry, whereas protein footprinting suggested a contribution from α-helix I residues Cys-280 and Lys-281. To more thoroughly characterize the vinylogous urea binding site, horizontal alanine scanning mutagenesis between p51 residues Lys-275 and Thr-286 (comprising α-helix I and portions of the neighboring αH/αI and αI/αJ connecting loops) was combined with a limited vertical scan of Cys-280. A contribution from Cys-280 was strengthened by our observation that all substitutions at this position rendered selectively mutated, reconstituted p66/p51 heterodimers ∼45-fold less sensitive to inhibition. An ∼19-fold reduced IC(50) for p51 mutant T286A coupled with a 2-8-fold increased IC(50) when intervening residues were substituted supports our original proposal of p51 α-helix I as the vinylogous urea binding site. In contrast to these allosteric inhibitors, mutant enzymes retained equivalent sensitivity to the natural product α-hydroxytropolone inhibitor manicol, which x-ray crystallography has demonstrated functions by chelating divalent metal at the p66 RNase H active site. Finally, reduced DNA strand-transfer activity together with increased vinylogous urea sensitivity of p66/p51 heterodimers containing short p51 C-terminal deletions suggests an additional role for the p51 C terminus in nucleic acid binding that is compromised by inhibitor binding.

  13. Congenital Short QT Syndrome

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    Johnson Francis

    2016-01-01

    Full Text Available Congenital short QT syndrome (SQTS is characterised by extremely short QT intervals, typically with QTc less than 330 ms and a propensity for life threatening ventricular arrhythmias and atrial fibrillation. The QT interval in SQTS does not change significantly with heart rate and the T waves have a narrow base and high voltage, similar to those in hyperkalemia.

  14. Idiopathic short stature

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    Vlaški Jovan

    2013-01-01

    Full Text Available Growth is a complex process and the basic characteristic of child- hood growth monitoring provides insight into the physiological and pathological events in the body. Statistically, the short stature means departure from the values of height for age and sex (in a particular environment, which is below -2 standard deviation score, or less than -2 standard deviation, i.e. below the third percentile. Advances in molecular genetics have contributed to the improvement of diagnostics in endocrinology. Analysis of patients’ genotypes should not be performed before taking a classical history, detailed clinical examination and appropriate tests. In patients with idiopathic short stature specific causes are excluded, such as growth hormone deficiency, Turner syndrome, short stature due to low birth weight, intrauterine growth retardation, small for gestational age, dysmorphology syndromes and chronic childhood diseases. The exclusion of abovementioned conditions leaves a large number of children with short stature whose etiology includes patients with genetic short stature or familial short stature and those who are low in relation to genetic potential, and who could also have some unrecognized endocrine defect. Idiopathic short stature represents a short stature of unknown cause of heterogeneous etiology, and is characterized by a normal response of growth hormone during stimulation tests (>10 ng/ml or 20 mJ/l, without other disorders, of normal body mass and length at birth. In idiopathic short stature standard deviation score rates <-2.25 (-2 to -3 or <1.2 percentile. These are also criteria for the initiation of growth hormone therapy. In children with short stature there is also the presence of psychological and social suffering. Goals of treatment with growth hormone involve achieving normal height and normal growth rate during childhood.

  15. Bidirectional promoter trapping T-DNA for insertional mutagenesis in Verticillium dahliae.

    Science.gov (United States)

    Deng, Sheng; Wang, Cai-yue; Zhang, Xin; Lin, Ling

    2014-07-01

    Transfer DNA (T-DNA)-based random insertional mutagenesis is a universal forward genetic approach for gene identification and cloning in many phytopathogenic fungi. In a large number of randomly selected transformants, screening for mutants with a specific phenotype is laborious, especially for pathogenicity-defective mutants. To accelerate mutant screening and gene identification, a bidirectional promoter-trapping Ti binary vector, 1300-bisGFP-hyg, was constructed and deployed in this study. More than 6000 Verticillium dahliae transformants were obtained by the mediation of Agrobacterium tumefaciens carrying the vector. One thousand randomly selected transformants were cultured on Czapek-Dox and on Czapek-Dox plus cotton root extract media plates. The cultured transformants with green fluorescent protein (GFP) expression or changes in phenotype were selected and used in virulence or promoter-trapping assays. Based on the virulence assay of 60 transformants, the pathogenicity of 17 of these mutants was compromised. Ten pathogenicity-defective mutants were found with GFP expression, and 6 with expression in Czapek-Dox plus cotton root extract media specifically. Using TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction), the T-DNA insertion sites were identified in 8 GFP-expressing transformants, including 5 pathogenicity-defective mutants and 3 unaffected transformants. Promoters of 6 genes were successfully trapped using the T-DNA method in this study. The nonpathogenic transformant 24C9 was the subject of additional investigation. It displayed strong GFP expression on water agar medium supplemented with cotton root extracts and on cotton seedling stems. The results obtained by Southern blot and quantitative real-time PCR confirmed that the transcription level of VdUGPU (encoding UTP-glucose-1-phosphate uridylyltransferase) was significantly reduced owing to T-DNA insertion in the gene promoter region. These results indicate that the bidirectional

  16. Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis.

    Science.gov (United States)

    DeJesus, Michael A; Gerrick, Elias R; Xu, Weizhen; Park, Sae Woong; Long, Jarukit E; Boutte, Cara C; Rubin, Eric J; Schnappinger, Dirk; Ehrt, Sabine; Fortune, Sarah M; Sassetti, Christopher M; Ioerger, Thomas R

    2017-01-17

    For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria. Sequencing of transposon-insertion mutant libraries has become a widely used tool for probing the functions of genes under various conditions. The Himar1 transposon is generally believed to insert with equal probabilities at all TA dinucleotides, and therefore its absence in a mutant library is taken to indicate biological selection against the corresponding mutant

  17. Insertional mutagenesis enables cleistothecial formation in a non-mating strain of Histoplasma capsulatum

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    Laskowski Meggan C

    2010-02-01

    Full Text Available Abstract Background Histoplasma capsulatum is a pathogenic ascomycete fungus that rapidly loses mating ability in culture. Loss of mating ability, as well as the organism's low rate of targeted gene replacement, limits techniques available for genetic studies in H. capsulatum. Understanding molecular mechanisms regulating mating in this organism may allow us to reverse or prevent loss of mating in H. capsulatum strains, introducing a variety of classical genetics techniques to the field. We generated a strain, UC1, by insertional mutagenesis of the laboratory strain G217B, and found that UC1 acquired the ability to form mating structures called cleistothecia. The aim of this study was to determine the mechanism by which UC1 gained the ability to form cleistothecia. We also present initial studies demonstrating that UC1 can be used as a tool to determine molecular correlates of mating in H. capsulatum. Results The strain UC1 was found to have increased RNA levels of the mating locus transcription factor (MAT1-1-1, and the putative alpha pheromone (PPG1 compared to G217B. Agrobacterium-mediated transformation and integration of T-DNA from the vector pCB301-GFP-HYG were found to be partially responsible for the increased RNA levels of these genes; however, the site of integration appeared to play the largest role in the strain's ability to form cleistothecia. Silencing HMK1, a putative FUS3/KSS1 homolog, had no effect on cleistothecial production by UC1. Protein kinase C (PKC1 RNA and protein levels were increased in UC1 compared to G217B, and pheromone production was found to be linked with Pkc1 activity in H. capsulatum. Conclusions The site of the T-DNA integration event appears to play the largest role in UC1's ability to form cleistothecia. We show that the UC1 strain can be used as a tool to study cleistothecia production in H. capsulatum by manipulating the strain, or by identifying differences between UC1 and G217B. Using these approaches

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

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    Hughes Lori A

    2005-11-01

    Full Text Available Abstract Background Analysis of an allelic series of point mutations in a gene, generated by N-ethyl-N-nitrosourea (ENU mutagenesis, is a valuable method for discovering the full scope of its biological function. Here we present an efficient gene-driven approach for identifying ENU-induced point mutations in any gene in C57BL/6J mice. The advantage of such an approach is that it allows one to select any gene of interest in the mouse genome and to go directly from DNA sequence to mutant mice. Results We produced the Cryopreserved Mutant Mouse Bank (CMMB, which is an archive of DNA, cDNA, tissues, and sperm from 4,000 G1 male offspring of ENU-treated C57BL/6J males mated to untreated C57BL/6J females. Each mouse in the CMMB carries a large number of random heterozygous point mutations throughout the genome. High-throughput Temperature Gradient Capillary Electrophoresis (TGCE was employed to perform a 32-Mbp sequence-driven screen for mutations in 38 PCR amplicons from 11 genes in DNA and/or cDNA from the CMMB mice. DNA sequence analysis of heteroduplex-forming amplicons identified by TGCE revealed 22 mutations in 10 genes for an overall mutation frequency of 1 in 1.45 Mbp. All 22 mutations are single base pair substitutions, and nine of them (41% result in nonconservative amino acid substitutions. Intracytoplasmic sperm injection (ICSI of cryopreserved spermatozoa into B6D2F1 or C57BL/6J ova was used to recover mutant mice for nine of the mutations to date. Conclusions The inbred C57BL/6J CMMB, together with TGCE mutation screening and ICSI for the recovery of mutant mice, represents a valuable gene-driven approach for the functional annotation of the mammalian genome and for the generation of mouse models of human genetic diseases. The ability of ENU to induce mutations that cause various types of changes in proteins will provide additional insights into the functions of mammalian proteins that may not be detectable by knockout mutations.

  19. Implementation of a loss-of-function system to determine growth and stress-associated mutagenesis in Bacillus subtilis.

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    Norberto Villegas-Negrete

    Full Text Available A forward mutagenesis system based on the acquisition of mutations that inactivate the thymidylate synthase gene (TMS and confer a trimethoprim resistant (Tmpr phenotype was developed and utilized to study transcription-mediated mutagenesis (TMM. In addition to thyA, Bacillus subtilis possesses thyB, whose expression occurs under conditions of cell stress; therefore, we generated a thyB- thyA+ mutant strain. Tmpr colonies of this strain were produced with a spontaneous mutation frequency of ~1.4 × 10-9. Genetic disruption of the canonical mismatch (MMR and guanine oxidized (GO repair pathways increased the Tmpr frequency of mutation by ~2-3 orders of magnitude. A wide spectrum of base substitutions as well as insertion and deletions in the ORF of thyA were found to confer a Tmpr phenotype. Stationary-phase-associated mutagenesis (SPM assays revealed that colonies with a Tmpr phenotype, accumulated over a period of ten days with a frequency of ~ 60 ×10-7. The Tmpr system was further modified to study TMM by constructing a ΔthyA ΔthyB strain carrying an IPTG-inducible Pspac-thyA cassette. In conditions of transcriptional induction of thyA, the generation of Tmpr colonies increased ~3-fold compared to conditions of transcriptional repression. Further, the Mfd and GreA factors were necessary for the generation of Tmpr colonies in the presence of IPTG in B. subtilis. Because GreA and Mfd facilitate transcription-coupled repair, our results suggest that TMM is a mechanim to produce genetic diversity in highly transcribed regions in growth-limited B. subtilis cells.

  20. An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.

    Science.gov (United States)

    Char, Si Nian; Neelakandan, Anjanasree K; Nahampun, Hartinio; Frame, Bronwyn; Main, Marcy; Spalding, Martin H; Becraft, Philip W; Meyers, Blake C; Walbot, Virginia; Wang, Kan; Yang, Bing

    2017-02-01

    CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium-delivered CRISPR/Cas9 for high-frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4). T 0 transgenic events carrying mono- or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi-II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T 1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target-specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  1. Site-directed in vitro immunization leads to a complete human monoclonal IgG4λ that binds specifically to the CDR2 region of CTLA-4 (CD152 without interfering the engagement of natural ligands

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    Hsu Shu-Ching

    2007-08-01

    Full Text Available Abstract Background The ability to acquire fully human monoclonal antibodies (mAbs with pre-defined specificities is critical to the development of molecular tags for the analysis of receptor function in addition to promising immunotherapeutics. Yet most of the arriving affinity maturated and complete human immunoglobulin G (IgG molecules, which are actually derived from single human B cells, have not widely been used to study the conserved self antigens (Ags such as CD152 (cytotoxic T lymphocyte antigen-4, CTLA-4 because proper hosts are lacking. Results Here we developed an optimized protocol for site-directed in vitro immunizing peripheral blood mononuclear cells (PBMC by using a selected epitope of human CD152, an essential receptor involved in down-regulation of T cell activation. The resultant stable trioma cell lines constantly produce anti-CD152 mAb (γ4λhuCD152, which contains variable (V regions of the heavy chain and the light chain derived from the VH3 and Vλ human germline genes, respectively, and yet displays an unusual IgG4 isotype. Interestingly, γ4λhuCD152 has a basic pI not commonly found in myeloid monoclonal IgG4λs as revealed by the isoelectric focusing (IEF analysis. Furthermore, γ4λhuCD152 binds specifically, with nanomolar affinity, to an extracellular constituency encompassing the putative second complementarity determining region (CDR2 of CD152, whereby it can react to activated CD3+ cells. Conclusion In a context of specific cell depletion and conditioned medium,in vitro induction of human Abs against a conserved self Ag was successfully acquired and a relatively basic mAb, γ4λhuCD152, with high affinity to CDR2 of CD152 was thus obtained. Application of such a human IgG4λ mAb with designated CDR2 specificity may impact upon and prefer for CD152 labeling both in situ and ex situ, as it does not affect the binding of endogenous B7 ligands and can localize into the confined immunological synapse which may

  2. Quest of novel GH20 N-acetyl hexosaminidasetransglycosylating catalysts: functional screening, data mining and semi-rational mutagenesis

    DEFF Research Database (Denmark)

    Teze, David; Visnapuu, Triinu; Kjeldsen, Christian

    and the fact that the products are also substrates, thus needing a kinetic control of the reaction. Several approaches have been developed to overcome these, including mechanism modifications (e.g. glycosynthases, chemical rescue), functional screening and data mining to find natural transglycosidases...... been reported. Thus, we turned to discovery and characterization of new GH20s and performing a systematic mutagenesis study. Several new GH20s of bacterial origin were isolated and described by functional screening and data mining, including transglycosidases able to synthesize lacto...

  3. Random mutagenesis of Luciola mingrelica firefly luciferase. Mutant enzymes with bioluminescence spectra showing low pH sensitivity.

    Science.gov (United States)

    Koksharov, M I; Ugarova, N N

    2008-08-01

    Most firefly luciferases demonstrate a strong pH-dependence of bioluminescence spectra. Gene region encoding first 225 residues of Luciola mingrelica luciferase was subjected to random mutagenesis, and four mutants with altered pH-sensitivity of bioluminescence spectra were isolated. F16L substitution showed distinctly lower pH-dependence of bioluminescence spectra, and Y35N,H and F16L/A40S substitutions resulted in the enzymes with bioluminescence spectra virtually independent from pH in the range of 6.0-7.8. The structural explanation is proposed for the effect of mutations on pH-sensitivity of bioluminescence spectra.

  4. Chemical mutagenesis of Gluconobacter frateurii to construct methanol-resistant mutants showing glyceric acid production from methanol-containing glycerol.

    Science.gov (United States)

    Sato, Shun; Kitamoto, Dai; Habe, Hiroshi

    2014-02-01

    To produce glyceric acid (GA) from methanol-containing glycerol, resistance to methanol of Gluconobacter frateurii NBRC103465 was improved by chemical mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine. The obtained mutant Gf398 produced 6.3 g/L GA in 5% (v/v) methanol-containing 17% (w/v) glycerol medium, in which the wild-type strain neither grew nor produced GA. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. Imaging in short stature

    Directory of Open Access Journals (Sweden)

    Vikas Chaudhary

    2012-01-01

    Full Text Available Short stature can be a sign of disease, disability, and social stigma causing psychological stress. It is important to have an early diagnosis and treatment. Short stature may result from skeletal dysplasias, endocrine disorders, may be familial, or may be the result of malnutrition and chronic illnesses. A team effort of the healthcare professionals like pediatricians, endocrinologists, radiologists, and pathologists is required to diagnose, treat and monitor various pathological conditions associated with growth abnormality. In this review, we have discussed the role of imaging in diagnosing and characterizing various pathological conditions associated with short stature.

  6. Improvement of the Chinese bean [Vigna Unguiculata (L.) Walp.], through radioinduced mutagenesis; Mejoramiento de Frijol Chino [Vigna Unguiculata (L.) Walp.], Mediante Mutagenesis Radioinducida

    Energy Technology Data Exchange (ETDEWEB)

    Salmeron E, J.; Bueno J, J.E.; Valencia E, F.; Solis M, M. [Colegio Superior Agropecuario del Estado de Guerrero, Iguala (Mexico); Cervantes S, T. [Instituto de Recursos Geneticos y Productividad (Mexico); Cruz T, E. de la [ININ, Carretera Mexico-La Marquesa S/N, La Marquesa Ocoyoacac, Mexico. C.P. 52750 (Mexico)]. e-mail: csaegro@prodigy.net.mx

    2006-07-01

    The advances in the process of genetic improvement of the Chinese bean (Vigna Unguiculata (L.) are presented, high nutritious value that it is evaluating as alternative for marginal areas producers of the State of Guerrero. The method of improvement applied it is recurrent radiation, continued by selection cycles applying the method of progeny by plant. The applied radiation doses were 200 and 250 Gray. The established selection approaches are: resistant plants or tolerant to the plagues attack and illnesses, vigorous, with more height to the first sheath, of compact and certain growth, with short internodes, bigger number of sheaths by plant and of grains by sheath, bigger number of grain size, among others. The obtained results show that the dose that induces bigger variability and that it has propitiated the biggest quantity in possible mutants it is 200Gy. Precocious plants with more height to the first sheath, with certain growth as well as with bigger number and sheaths size have been detected. The selected plants have incorporated to an increment process by means of the progeny method by plant. (Author)

  7. A short note on short pants

    OpenAIRE

    Parlier, Hugo

    2015-01-01

    It is a theorem of Bers that any closed hyperbolic surface admits a pants decomposition consisting of curves of bounded length where the bound only depends on the topology of the surface. The question of the quantification of the optimal constants has been well studied and the best upper bounds to date are linear in genus, a theorem of Buser and Sepp\\"al\\"a. The goal of this note is to give a short proof of an linear upper bound which slightly improves the best known bounds.

  8. Description of a PCR-based technique for DNA splicing and mutagenesis by producing 5' overhangs with run through stop DNA synthesis utilizing Ara-C

    Directory of Open Access Journals (Sweden)

    Silverman Mel

    2005-09-01

    Full Text Available Abstract Background Splicing of DNA molecules is an important task in molecular biology that facilitates cloning, mutagenesis and creation of chimeric genes. Mutagenesis and DNA splicing techniques exist, some requiring restriction enzymes, and others utilize staggered reannealing approaches. Results A method for DNA splicing and mutagenesis without restriction enzymes is described. The method is based on mild template-dependent polymerization arrest with two molecules of cytosine arabinose (Ara-C incorporated into PCR primers. Two rounds of PCR are employed: the first PCR produces 5' overhangs that are utilized for DNA splicing. The second PCR is based on polymerization running through the Ara-C molecules to produce the desired final product. To illustrate application of the run through stop mutagenesis and DNA splicing technique, we have carried out splicing of two segments of the human cofilin 1 gene and introduced a mutational deletion into the product. Conclusion We have demonstrated the utility of a new PCR-based method for carrying out DNA splicing and mutagenesis by incorporating Ara-C into the PCR primers.

  9. A Practical Strategy to Discover New Antitumor Compounds by Activating Silent Metabolite Production in Fungi by Diethyl Sulphate Mutagenesis

    Directory of Open Access Journals (Sweden)

    Shi-Ming Fang

    2014-03-01

    Full Text Available Many fungal biosynthetic pathways are silent in standard culture conditions, and activation of the silent pathways may enable access to new metabolites with antitumor activities. The aim of the present study was to develop a practical strategy for microbial chemists to access silent metabolites in fungi. We demonstrated this strategy using a marine-derived fungus Penicillium purpurogenum G59 and a modified diethyl sulphate mutagenesis procedure. Using this strategy, we discovered four new antitumor compounds named penicimutanolone (1, penicimutanin A (2, penicimutanin B (3, and penicimutatin (4. Structures of the new compounds were elucidated by spectroscopic methods, especially extensive 2D NMR analysis. Antitumor activities were assayed by the MTT method using human cancer cell lines. Bioassays and HPLC-photodiode array detector (PDAD-UV and HPLC-electron spray ionization (ESI-MS analyses were used to estimate the activated secondary metabolite production. Compounds 2 and 3 had novel structures, and 1 was a new compound belonging to a class of very rare natural products from which only four members are so far known. Compounds 1–3 inhibited several human cancer cell lines with IC50 values lower than 20 μM, and 4 inhibited the cell lines to some extent. These results demonstrated the effectiveness of this strategy to discover new compounds by activating silent fungal metabolic pathways. These discoveries provide rationale for the increased use of chemical mutagenesis strategies in silent fungal metabolite studies.

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

    Science.gov (United States)

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

    2013-01-01

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

  11. Effects of vitamins A and E on methylazoxymethanol-induced mutagenesis in Salmonella typhimurium strain TA100.

    Science.gov (United States)

    Tavan, E; Maziere, S; Narbonne, J F; Cassand, P

    1997-07-03

    The aim of this study is to report the antimutagenic effect of vitamin A and vitamin E towards methylazoxymethanol (MAM)-induced mutagenesis in Salmonella typhimurium strain TA100 sensitive to alkylating agents. In order to characterize different levels of action of these two fat-soluble vitamins towards the mutagenicity of MAM, several assays have been considered to show the antimutagenic effect and the possible interactions of vitamins with MAM or with the bacteria. Thus, for each vitamin, three different assays with three different incubations have been conducted: (i) MAM, bacteria and vitamins together, (ii) MAM and vitamins, (iii) bacteria and vitamins. The results showed that both vitamins A and E present an antimutagenic effect towards MAM induced mutagenesis. alpha-Tocopherol seems to have an action directly on to the mutagenic agent, whereas the action of retinol is likely due to a protection of the bacterial genoma against MAM. These in vitro results could help to interpret results of colon carcinogenesis studies using animals induced by 1,2-dimethylhydrazine and fed vitamins supplemented diet.

  12. Mutation breeding of extracellular polysaccharide-producing microalga Crypthecodinium cohnii by a novel mutagenesis with atmospheric and room temperature plasma.

    Science.gov (United States)

    Liu, Bin; Sun, Zheng; Ma, Xiaonian; Yang, Bo; Jiang, Yue; Wei, Dong; Chen, Feng

    2015-04-13

    Extracellular polysaccharides (EPS) produced by marine microalgae have the potential to be used as antioxidants, antiviral agents, immunomodulators, and anti-inflammatory agents. Although the marine microalga Crypthecodinium cohnii releases EPS during the process of docosahexaenoic acid (DHA) production, the yield of EPS remains relatively low. To improve the EPS production, a novel mutagenesis of C. cohnii was conducted by atmospheric and room temperature plasma (ARTP). Of the 12 mutants obtained, 10 mutants exhibited significantly enhanced EPS yield on biomass as compared with the wild type strain. Among them, mutant M7 was the best as it could produce an EPS volumetric yield of 1.02 g/L, EPS yield on biomass of 0.39 g/g and EPS yield on glucose of 94 mg/g, which were 33.85%, 85.35% and 57.17% higher than that of the wild type strain, respectively. Results of the present study indicated that mutagenesis of the marine microalga C. cohnii by ARTP was highly effective leading to the high-yield production of EPS.

  13. Tailoring of global transcription sigma D factor by random mutagenesis to improve Escherichia coli tolerance towards low-pHs.

    Science.gov (United States)

    Gao, Xi; Jiang, Ling; Zhu, Liying; Xu, Qing; Xu, Xian; Huang, He

    2016-04-20

    Bioconversion processes of organic acid or acid hydrolysis of raw material for microbial metabolism often suffer limitations as a result of microbial sensitivity in low-pH conditions. We adopted a three-step method called RAndom Insertional-deletional Strand Exchange mutagenesis (RAISE) to engineer the components of global regulator Sigma D factor (RpoD) of Escherichia coli to improve its acid tolerance. The best strain Mutant VII was identified from random mutagenesis libraries based on the growth performance, which exhibited much higher growth rate than the control (0.22h(-1) vs. 0.15h(-1)) at pH as low as 3.17. Combined transcriptome and phenome analysis of E. coli was carried out to better understand the global effects of RpoD on the regulatory networks. Our analysis showed that 95 (2.1%) of all E. coli genes were induced and 178 (4.0%) genes were repressed, including those for trehalose biosynthesis, nucleotides biosynthesis, carbon metabolism, amino acid utilization, except for acid resistance. Also regulated were the master regulators (ArcA, EvgA, H-NS and RpoS) and gene/operon-specific transcription factors (GadX, GadW, AppY, YdeO, KdgR). These results demonstrated that RpoD acts as global regulator in the growth phase of E. coli and consequently improves acid tolerances. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Mutation Breeding of Extracellular Polysaccharide-Producing Microalga Crypthecodinium cohnii by a Novel Mutagenesis with Atmospheric and Room Temperature Plasma

    Directory of Open Access Journals (Sweden)

    Bin Liu

    2015-04-01

    Full Text Available Extracellular polysaccharides (EPS produced by marine microalgae have the potential to be used as antioxidants, antiviral agents, immunomodulators, and anti-inflammatory agents. Although the marine microalga Crypthecodinium cohnii releases EPS during the process of docosahexaenoic acid (DHA production, the yield of EPS remains relatively low. To improve the EPS production, a novel mutagenesis of C. cohnii was conducted by atmospheric and room temperature plasma (ARTP. Of the 12 mutants obtained, 10 mutants exhibited significantly enhanced EPS yield on biomass as compared with the wild type strain. Among them, mutant M7 was the best as it could produce an EPS volumetric yield of 1.02 g/L, EPS yield on biomass of 0.39 g/g and EPS yield on glucose of 94 mg/g, which were 33.85%, 85.35% and 57.17% higher than that of the wild type strain, respectively. Results of the present study indicated that mutagenesis of the marine microalga C. cohnii by ARTP was highly effective leading to the high-yield production of EPS.

  15. High efficiency of targeted mutagenesis in arabidopsis via meiotic promoter-driven expression of Cas9 endonuclease

    KAUST Repository

    Eid, Ayman

    2016-05-28

    Key message: The use of a meiosis I-specific promoter increased the efficiency of targeted mutagenesis and will facilitate the manipulation of homologous recombination. Abstract: The CRISPR/Cas9 system has been harnessed for targeted engineering of eukaryotic genomes, including plants; however, CRISPR/Cas9 efficiency varies considerably in different plant tissues and species. In Arabidopsis, the generation of homozygous or bi-allelic mutants in the first (T1) generation is inefficient. Here, we used specific promoters to drive the expression of Cas9 during meiosis to maximize the efficiency of recovering heritable mutants in T1 plants. Our data reveal that the use of a promoter active in meiosis I resulted in high-efficiency (28 %) recovery of targeted mutants in the T1 generation. Moreover, this method enabled efficient simultaneous targeting of three genes for mutagenesis. Taken together, our results show that the use of meiosis-specific promoters will improve methods for functional genomic analysis and studying the molecular underpinnings of homologous recombination. © 2016, Springer-Verlag Berlin Heidelberg.

  16. Radiation-induced in vitro mutagenesis system for salt tolerance and other agronomic characters in sugarcane (Saccharum officinarum L.

    Directory of Open Access Journals (Sweden)

    Ashok A. Nikam

    2015-02-01

    Full Text Available Gamma ray-induced in vitro mutagenesis and selection for salt (NaCl tolerance were investigated in sugarcane (Saccharum officinarum L.. Embryogenic callus cultures were irradiated (10 to 80 Gy and subjected to in vitro selection by exposure of irradiated callus to NaCl (0, 50, 100, 150, 200, and 250 mmol L− 1. Increasing NaCl concentrations resulted in growth reduction and increased membrane damage. Salt-selected callus lines were characterized by the accumulation of proline, glycine betaine, and Na+ and K+ concentration. Higher accumulation of proline and glycine betaine was observed in NaCl stressed callus irradiated at 20 Gy. Na+ concentration increased and K+ concentration decreased with increasing salt level. Irradiated callus showed 50–60% regeneration under NaCl stress, and in vitro-regenerated plants were acclimatized in the greenhouse, with 80–85% survival. A total of 138 irradiated and salt-selected selections were grown to maturity and their agronomic performance was evaluated under normal and saline conditions. Of these, 18 mutant clones were characterized for different agro-morphological characters and some of the mutant clones exhibited improved sugar yield with increased Brix%, number of millable canes, and yield. The result suggest that radiation-induced mutagenesis offers an effective way to enhance genetic variation in sugarcane.

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

    Science.gov (United States)

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

    2010-05-01

    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.

  18. Targeted mutagenesis of the Clostridium acetobutylicum acetone-butanol-ethanol fermentation pathway.

    Science.gov (United States)

    Cooksley, Clare M; Zhang, Ying; Wang, Hengzheng; Redl, Stephanie; Winzer, Klaus; Minton, Nigel P

    2012-11-01

    The production of the chemical solvents acetone and butanol by the bacterium Clostridium acetobutylicum was one of the first large-scale industrial processes to be developed, and in the first part of the last century ranked second in importance only to ethanol production. After a steep decline in its industrial use, there has been a recent resurgence of interest in the acetone-butanol-ethanol (ABE) fermentation process, with a particular emphasis on butanol production. In order to generate strains suitable for efficient use on an industrial scale, metabolic engineering is required to alter the AB ratio in favour of butanol, and eradicate the production of unwanted products of fermentation. Using ClosTron technology, a large-scale targeted mutagenesis in C. acetobutylicum ATCC 824 was carried out, generating a set of 10 mutants, defective in alcohol/aldehyde dehydrogenases 1 and 2 (adhE1, adhE2), butanol dehydrogenases A and B (bdhA, bdhB), phosphotransbutyrylase (ptb), acetate kinase (ack), acetoacetate decarboxylase (adc), CoA transferase (ctfA/ctfB), and a previously uncharacterised putative alcohol dehydrogenase (CAP0059). However, inactivation of the main hydrogenase (hydA) and thiolase (thl) could not be achieved. Constructing such a series of mutants is paramount for the acquisition of information on the mechanism of solvent production in this organism, and the subsequent development of industrial solvent producing strains. Unexpectedly, bdhA and bdhB mutants did not affect solvent production, whereas inactivation of the previously uncharacterised gene CAP0059 resulted in increased acetone, butanol, and ethanol formation. Other mutants showed predicted phenotypes, including a lack of acetone formation (adc, ctfA, and ctfB mutants), an inability to take up acids (ctfA and ctfB mutants), and a much reduced acetate formation (ack mutant). The adhE1 mutant in particular produced very little solvents, demonstrating that this gene was indeed the main contributor to

  19. Congenital Short QT Syndrome

    Directory of Open Access Journals (Sweden)

    Lia Crotti

    2010-02-01

    Full Text Available The Short QT Syndrome is a recently described new genetic disorder, characterized by abnormally short QT interval, paroxysmal atrial fibrillation and life threatening ventricular arrhythmias. This autosomal dominant syndrome can afflict infants, children, or young adults; often a remarkable family background of cardiac sudden death is elucidated. At electrophysiological study, short atrial and ventricular refractory periods are found, with atrial fibrillation and polymorphic ventricular tachycardia easily induced by programmed electrical stimulation. Gain of function mutations in three genes encoding K+ channels have been identified, explaining the abbreviated repolarization seen in this condition: KCNH2 for Ikr (SQT1, KCNQ1 for Iks (SQT2 and KCNJ2 for Ik1 (SQT3. The currently suggested therapeutic strategy is an ICD implantation, although many concerns exist for asymptomatic patients, especially in pediatric age. Pharmacological treatment is still under evaluation; quinidine has shown to prolong QT and reduce the inducibility of ventricular arrhythmias, but awaits additional confirmatory clinical data.

  20. Short wavelength FELS

    Energy Technology Data Exchange (ETDEWEB)

    Sheffield, R.L.

    1991-01-01

    The generation of coherent ultraviolet and shorter wavelength light is presently limited to synchrotron sources. The recent progress in the development of brighter electron beams enables the use of much lower energy electron rf linacs to reach short-wavelengths than previously considered possible. This paper will summarize the present results obtained with synchrotron sources, review proposed short- wavelength FEL designs and then present a new design which is capable of over an order of magnitude higher power to the extreme ultraviolet. 17 refs., 10 figs.

  1. SHORT PULSE STRETCHER

    Science.gov (United States)

    Branum, D.R.; Cummins, W.F.

    1962-12-01

    >A short pulse stretching circuit capable of stretching a short puise to enable it to be displayed on a relatively slow sweeping oscilloscope is described. Moreover, the duration of the pulse is increased by charging a capacitor through a diode and thereafter discharging the capacitor at such time as is desired. In the circuit the trigger pulse alone passes through a delay line, whereas the main signal passes through the diode only, and results in over-all circuit losses which are proportional to the low losses of the diode only. (AEC)

  2. Research Short report

    African Journals Online (AJOL)

    knowledge and best practice were evaluated at the participating facilities, both pre and post intervention. Results. Most survey respondents (239/271; 88.2%) practised in rural districts and reportedly received infrequent (either annual or no) in-service training in IPC (138/271; 51%). The IPC education intervention (five short ...

  3. Shortness of Breath

    Science.gov (United States)

    ... with obesity hypoventilation syndrome also have sleep apnea. Deconditioning If you are not active or do not exer- cise regularly, as a result of being out of shape and experiencing muscle fatigue, you may develop shortness of breath with physical exertion beyond your customary activity such as when ...

  4. SHORT RIB POLYDACTYLY SYNDROME

    Directory of Open Access Journals (Sweden)

    Z Moinfar

    2007-09-01

    Full Text Available Short rib polydactyly syndrome (SRPS is a very rare congenital anomaly that is classified into four subtypes. It is an autosomal recessive inherited disease. We report a case of this syndrome without a previous family history of congenital defects.

  5. SHORT COMMUNICATION POLYCYCLIC AROMATIC ...

    African Journals Online (AJOL)

    a

    ______. *Corresponding author. E-mail: omotayosharafdeen@yahoo.com. SHORT COMMUNICATION. POLYCYCLIC AROMATIC HYDROCARBONS IN MUNICIPAL WASTE ASHES. FROM THREE WASTE DUMPS IN LAGOS, NIGERIA. O.S. Amuda* and F.E. Adelowo-Imeokparia. Environmental/Analytical Chemistry Unit, ...

  6. Congenital Short QT Syndrome

    Directory of Open Access Journals (Sweden)

    Charles Antzelevitch

    2004-04-01

    Full Text Available Long QT intervals in the ECG have long been associated with sudden cardiac death. The congenital long QT syndrome was first described in individuals with structurally normal hearts in 1957.1 Little was known about the significance of a short QT interval. In 1993, after analyzing 6693 consecutive Holter recordings Algra et al concluded that an increased risk of sudden death was present not only in patients with long QT interval, but also in patients with short QT interval (<400 ms.2 Because this was a retrospective analysis, further evaluation of the data was not possible. It was not until 2000 that a short-QT syndrome (SQTS was proposed as a new inherited clinical syndrome by Gussak et al.3 The initial report was of two siblings and their mother all of whom displayed persistently short QT interval. The youngest was a 17 year old female presenting with several episodes of paroxysmal atrial fibrillation requiring electrical cardioversion.3 Her QT interval measured 280 msec at a heart rate of 69. Her 21 year old brother displayed a QT interval of 272 msec at a heart rate of 58, whereas the 51 year old mother showed a QT of 260 msec at a heart rate of 74. The authors also noted similar ECG findings in another unrelated 37 year old patient associated with sudden cardiac death.

  7. Ultra-wide band electromagnetic radiation does not affect UV-induced recombination and mutagenesis in yeast.

    Science.gov (United States)

    Pakhomova, O N; Belt, M L; Mathur, S P; Lee, J C; Akyel, Y

    1998-01-01

    Cell samples of the yeast Saccharomyces cerevisiae were exposed to 100 J/m2 of 254 nm ultraviolet (UV) radiation followed by a 30 min treatment with ultra-wide band (UWB) electromagnetic pulses. The UWB pulses (101-104 kV/m, 1.0 ns width, 165 ps rise time) were applied at the repetition rates of 0 Hz (sham), 16 Hz, or 600 Hz. The effect of exposures was evaluated from the colony-forming ability of the cells on complete and selective media and the number of aberrant colonies. The experiments established no effect of UWB exposure on the UV-induced reciprocal and non-reciprocal recombination, mutagenesis, or cell survival.

  8. The antimutagenic effect of monoterpenes against UV-irradiation-, 4NQO- and t-BOOH-induced mutagenesis in coli

    Directory of Open Access Journals (Sweden)

    Nikolić Biljana

    2011-01-01

    Full Text Available The aim of this work was to investigate the antimutagenic potential of monoterpenes from sage and basil in Escherichia coli. The mutagenic potential of monoterpenes was pre-screened with Salmonella/microsome reversion assay in strain TA100 and no mutagenic effect was detected. The antimutagenic potential against UV- 4NQO- and t-BOOH induced mutagenesis was evaluated in E. coli K12 and E. coli WP2 by reversion assays. The obtained results indicate that camphor and thujone reduce UV- and 4NQO-induced mutations; myrcene reduces t-BOOH-induced mutations, while eucalyptol and linalool reduce mutagenicity by all tested mutagens. Considering evolutionary conservation of DNA repair and antioxidative protection, the obtained results indicate that further antigenotoxicity studies should be undertaken in eukaryotes.

  9. Random mutagenesis MAPPIT analysis identifies binding sites for Vif and Gag in both cytidine deaminase domains of Apobec3G.

    Directory of Open Access Journals (Sweden)

    Isabel Uyttendaele

    Full Text Available The mammalian two-hybrid system MAPPIT allows the detection of protein-protein interactions in intact human cells. We developed a random mutagenesis screening strategy based on MAPPIT to detect mutations that disrupt the interaction of one protein with multiple protein interactors simultaneously. The strategy was used to detect residues of the human cytidine deaminase Apobec3G that are important for its homodimerization and its interaction with the HIV-1 Gag and Vif proteins. The strategy is able to identify the previously described head-to-head homodimerization interface in the N-terminal domain of Apobec3G. Our analysis further detects two new potential interaction surfaces in the N-and C-terminal domain of Apobec3G for interaction with Vif and Gag or for Apobec3G dimerization.

  10. Agrobacterium tumefaciens-mediated transformation as an efficient tool for insertional mutagenesis of Cercospora zeae-maydis.

    Science.gov (United States)

    Lu, Yuanyuan; Xiao, Shuqin; Wang, Fen; Sun, Jiaying; Zhao, Likun; Yan, Libin; Xue, Chunsheng

    2017-02-01

    An efficient Agrobacterium tumefaciens-mediated transformation (ATMT) approach was developed for the plant pathogenic fungus, Cercospora zeae-maydis, which is the causative agent of gray leaf spot in maize. The transformation was evaluated with five parameters to test the efficiencies of transformation. Results showed that spore germination time, co-cultivation temperature and time were the significant influencing factors in all parameters. Randomly selected transformants were confirmed and the transformants were found to be mitotically stable, with single-copy T-DNA integration in the genome. T-DNA flanking sequences were cloned by thermal asymmetric interlaced PCR. Thus, the ATMT approach is an efficient tool for insertional mutagenesis of C. zeae-maydis. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Signature-tagged mutagenesis of Pasteurella multocida identifies mutants displaying differential virulence characteristics in mice and chickens.

    Science.gov (United States)

    Harper, Marina; Boyce, John D; Wilkie, Ian W; Adler, Ben

    2003-09-01

    Pasteurella multocida is the causative agent of fowl cholera in birds. Signature-tagged mutagenesis (STM) was used to identify potential virulence factors in a mouse septicemia disease model and a chicken fowl cholera model. A library of P. multocida mutants was constructed with a modified Tn916 and screened for attenuation in both animal models. Mutants identified by the STM screening were confirmed as attenuated by competitive growth assays in both chickens and mice. Of the 15 mutants identified in the chicken model, only 5 were also attenuated in mice, showing for the first time the presence of host-specific virulence factors and indicating the importance of screening for attenuation in the natural host.

  12. Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis.

    Directory of Open Access Journals (Sweden)

    Feixiong Cheng

    2016-09-01

    Full Text Available Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase. Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline that may be potential for antiviral indication (e.g. anti-Ebola. In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics.

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

    Energy Technology Data Exchange (ETDEWEB)

    Mehta, Kinjal Rajesh; Chan, Yan M.; Lee, Man X.; Yang, Ching Yao; Voloshchuk, Natalya [Department of Chemical and Biological Sciences, Polytechnic Institute of New York University, 6 MetroTech Center, Brooklyn, NY 11201 (United States); Montclare, Jin Kim, E-mail: jmontcla@poly.edu [Department of Chemical and Biological Sciences, Polytechnic Institute of New York University, 6 MetroTech Center, Brooklyn, NY 11201 (United States); Department of Biochemistry, SUNY-Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203 (United States)

    2010-09-24

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

  14. Generation of peanut drought tolerant plants by pingyangmycin-mediated in vitro mutagenesis and hydroxyproline-resistance screening.

    Science.gov (United States)

    Sui, Jiongming; Wang, Ya; Wang, Peng; Qiao, Lixian; Sun, Shimeng; Hu, Xiaohui; Chen, Jing; Wang, Jingshan

    2015-01-01

    In order to enlarge the potential resources of drought-tolerant peanuts, we conducted in vitro mutagenesis with Pingyangmycin (PYM) as the mutagen as well as directed screening on a medium supplemented with Hydroxyproline (HYP). After being extracted from mature seeds (cv. Huayu 20), the embryonic leaflets were cultured on somatic embryogenesis-induction medium with 4 mg/L PYM and the generated embryos were successively transferred to a germination medium with 4 and then 8 mmol/L HYP to screen HYP-tolerant plantlets. After that, these plantlets were grafted and transplanted to the experimental field. In the next generation, all seeds were sown in the field, and phenotype variation and trait segregation can be observed in most of the offspring (M2 generation). The M3 generation individuals were subjected to drought stress at the seedling stages. The activities of SOD and POD were substantially increased in eight offspring of 11 HYP-tolerant, regenerated plants than in their mutagenic parents. To determine the correlation between mutant phenotypes and genomic modification, we carried out a comparison of the DNA polymorphisms between the mutagenic parents and 13 M3 generation individuals from different HYP-tolerant, regenerated plants with SSR primers. Results showed that most mutants and parent plants had signs of polymorphisms. Under drought stress, some M3 generation individuals of 10 original HYP-tolerant, regenerated plants produced more pods than the mutagenic parent; twenty individuals among them produced >60 g pods/plant. M4-generation seeds were tested for quality characteristics by Near Infrared Spectroscopy (NIS) and nine individuals with higher protein content (>30%) and 21 individuals with higher oil content (>58%) were screened. We concluded that the use of PYM-based in vitro mutagenesis in combination with directed screening with HYP is effective for the creation of potential drought-tolerant mutants of peanut.

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

    Science.gov (United States)

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

    2016-01-11

    Crustaceans possess a diverse array of specialized limbs. Although shifts in Hox gene expression domains have been postulated to play a role in generating this limb diversity, little functional data have been provided to understand the precise roles of Hox genes during crustacean development. We used a combination of CRISPR/Cas9-targeted mutagenesis and RNAi knockdown to decipher the function of the six Hox genes expressed in the developing mouth and trunk of the amphipod Parhyale hawaiensis. These experimentally manipulated animals display specific and striking homeotic transformations. We found that abdominal-A (abd-A) and Abdominal-B (Abd-B) are required for proper posterior patterning, with knockout of Abd-B resulting in an animal with thoracic type legs along what would have been an abdomen, and abd-A disruption generating a simplified body plan characterized by a loss of specialization in both abdominal and thoracic appendages. In the thorax, Ubx is necessary for gill development and for repression of gnathal fate, and Antp dictates claw morphology. In the mouth, Scr and Antp confer the part-gnathal, part-thoracic hybrid identity of the maxilliped, and Scr and Dfd prevent antennal identity in posterior head segments. Our results allow us to define the role Hox genes play in specifying each appendage type in Parhyale, including the modular nature by which some appendages are patterned by Hox gene inputs. In addition, we define how changes in Hox gene expression have generated morphological differences between crustacean species. Finally, we also highlight the utility of CRISPR/Cas9-based somatic mutagenesis in emerging model organisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. 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.

  17. Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion.

    Science.gov (United States)

    Fulton, Benjamin O; Sachs, David; Schwarz, Megan C; Palese, Peter; Evans, Matthew J

    2017-08-01

    The molecular constraints affecting Zika virus (ZIKV) evolution are not well understood. To investigate ZIKV genetic flexibility, we used transposon mutagenesis to add 15-nucleotide insertions throughout the ZIKV MR766 genome and subsequently deep sequenced the viable mutants. Few ZIKV insertion mutants replicated, which likely reflects a high degree of functional constraints on the genome. The NS1 gene exhibited distinct mutational tolerances at different stages of the screen. This result may define regions of the NS1 protein that are required for the different stages of the viral life cycle. The ZIKV structural genes showed the highest degree of insertional tolerance. Although the envelope (E) protein exhibited particular flexibility, the highly conserved envelope domain II (EDII) fusion loop of the E protein was intolerant of transposon insertions. The fusion loop is also a target of pan-flavivirus antibodies that are generated against other flaviviruses and neutralize a broad range of dengue virus and ZIKV isolates. The genetic restrictions identified within the epitopes in the EDII fusion loop likely explain the sequence and antigenic conservation of these regions in ZIKV and among multiple flaviviruses. Thus, our results provide insights into the genetic restrictions on ZIKV that may affect the evolution of this virus.IMPORTANCE Zika virus recently emerged as a significant human pathogen. Determining the genetic constraints on Zika virus is important for understanding the factors affecting viral evolution. We used a genome-wide transposon mutagenesis screen to identify where mutations were tolerated in replicating viruses. We found that the genetic regions involved in RNA replication were mostly intolerant of mutations. The genes coding for structural proteins were more permissive to mutations. Despite the flexibility observed in these regions, we found that epitopes bound by broadly reactive antibodies were genetically constrained. This finding may explain

  18. Generation of peanut drought tolerant plants by pingyangmycin-mediated in vitro mutagenesis and hydroxyproline-resistance screening.

    Directory of Open Access Journals (Sweden)

    Jiongming Sui

    Full Text Available In order to enlarge the potential resources of drought-tolerant peanuts, we conducted in vitro mutagenesis with Pingyangmycin (PYM as the mutagen as well as directed screening on a medium supplemented with Hydroxyproline (HYP. After being extracted from mature seeds (cv. Huayu 20, the embryonic leaflets were cultured on somatic embryogenesis-induction medium with 4 mg/L PYM and the generated embryos were successively transferred to a germination medium with 4 and then 8 mmol/L HYP to screen HYP-tolerant plantlets. After that, these plantlets were grafted and transplanted to the experimental field. In the next generation, all seeds were sown in the field, and phenotype variation and trait segregation can be observed in most of the offspring (M2 generation. The M3 generation individuals were subjected to drought stress at the seedling stages. The activities of SOD and POD were substantially increased in eight offspring of 11 HYP-tolerant, regenerated plants than in their mutagenic parents. To determine the correlation between mutant phenotypes and genomic modification, we carried out a comparison of the DNA polymorphisms between the mutagenic parents and 13 M3 generation individuals from different HYP-tolerant, regenerated plants with SSR primers. Results showed that most mutants and parent plants had signs of polymorphisms. Under drought stress, some M3 generation individuals of 10 original HYP-tolerant, regenerated plants produced more pods than the mutagenic parent; twenty individuals among them produced >60 g pods/plant. M4-generation seeds were tested for quality characteristics by Near Infrared Spectroscopy (NIS and nine individuals with higher protein content (>30% and 21 individuals with higher oil content (>58% were screened. We concluded that the use of PYM-based in vitro mutagenesis in combination with directed screening with HYP is effective for the creation of potential drought-tolerant mutants of peanut.

  19. Genomic saturation mutagenesis and polygenic analysis identify novel yeast genes affecting ethyl acetate production, a non-selectable polygenic trait

    Science.gov (United States)

    Abt, Tom Den; Souffriau, Ben; Foulquié-Moreno, Maria R.; Duitama, Jorge; Thevelein, Johan M.

    2016-01-01

    Isolation of mutants in populations of microorganisms has been a valuable tool in experimental genetics for decades. The main disadvantage, however, is the inability of isolating mutants in non-selectable polygenic traits. Most traits of organisms, however, are non-selectable and polygenic, including industrially important properties of microorganisms. The advent of powerful technologies for polygenic analysis of complex traits has allowed simultaneous identification of multiple causative mutations among many thousands of irrelevant mutations. We now show that this also applies to haploid strains of which the genome has been loaded with induced mutations so as to affect as many non-selectable, polygenic traits as possible. We have introduced about 900 mutations into single haploid yeast strains using multiple rounds of EMS mutagenesis, while maintaining the mating capacity required for genetic mapping. We screened the strains for defects in flavor production, an important non-selectable, polygenic trait in yeast alcoholic beverage production. A haploid strain with multiple induced mutations showing reduced ethyl acetate production in semi-anaerobic fermentation, was selected and the underlying quantitative trait loci (QTLs) were mapped using pooled-segregant whole-genome sequence analysis after crossing with an unrelated haploid strain. Reciprocal hemizygosity analysis and allele exchange identified PMA1 and CEM1 as causative mutant alleles and TPS1 as a causative genetic background allele. The case of CEM1 revealed that relevant mutations without observable effect in the haploid strain with multiple induced mutations (in this case due to defective mitochondria) can be identified by polygenic analysis as long as the mutations have an effect in part of the segregants (in this case those that regained fully functional mitochondria). Our results show that genomic saturation mutagenesis combined with complex trait polygenic analysis could be used successfully to

  20. Relationship of DNA repair processes to mutagenesis and carcinogenesis in mammalian cells. Progress report, November 1, 1979-October 31, 1980

    Energy Technology Data Exchange (ETDEWEB)

    Evans, H.H.

    1980-10-01

    The objective of this research is to determine the role of DNA repair in mutagenesis and carcinogenesis in mammalian cells. Use of the host-cell reactivation viral suicide enrichment procedure was initiated in the isolation of repair-deficient mutants. Lightly mutagenized BHK cells were infected with irradiated Herpes simplex virus (HSV); several radiation-sensitive strains were isolated among the survivors of the infection. The characterization of these strains is progressing and the enrichments are continuing. That alterations in the frequency of mutation of C3H/10T 1/2 cells, occurring as a result of holding the cells in a confluent state following treatment with ethylmethane sulfonate, parallel the alterations in the frequency of neoplastic transformation was found. The repair capabilities of BHK cells were found to be intermediate in comparison to repair-proficient and -deficient human cells with regard to the reactivation of HSV treated with various inactivating agents. The effect of confluency and of low serum levels on DNA synthesis, as well as the response to the cytotoxic effects of MNNG and acriflavin were determined in BHK cells in preparation for the investigation of the role of DNA repair in mutagenesis and transformation. It was also found that C3H/10T 1/2 cells partially recover from the toxic effects of 4-nitroquinoline-1-oxide if they are held in a confluent state for 6 to 22 hrs following treatment. Addition of catalase did not alleviate the toxic effects of 4-NQO. The cells contain a relatively high endogenous level of this enzyme. (ERB)