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Sample records for modeling site-directed mutagenesis

  1. Site-directed mutagenesis.

    Science.gov (United States)

    Bachman, Julia

    2013-01-01

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

  2. Progress and prospects: techniques for site-directed mutagenesis in animal models

    OpenAIRE

    Yan, Z.; Sun, X.; Engelhardt, JF

    2009-01-01

    In the past 2 years, new gene-targeting approaches using adeno-associated virus and designer zinc-finger nucleases have been successfully applied to the production of genetically modified ferrets, pigs, mice and zebrafish. Gene targeting using these tools has been combined with somatic cell nuclear transfer and germ cell transplantation to generate gene-targeted animal models. These new technical advances, which do not require the generation of embryonic stem cell-derived chimeras, will great...

  3. Efficient multi-site-directed mutagenesis directly from genomic template

    Indian Academy of Sciences (India)

    Fengtao Luo; Xiaolan Du; Tujun Weng; Xuan Wen; Junlan Huang; Lin Chen

    2012-12-01

    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 as for combination, deletion or insertion of DNA fragments.

  4. Analysis of mammalian cytochrome P450 structure and function by site-directed mutagenesis.

    Science.gov (United States)

    Domanski, T L; Halpert, J R

    2001-06-01

    Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology modeling, chimeragenesis, and site-directed mutagenesis. As homology modeling and substrate docking technology continue to improve, the ability to predict more precise functions for specific residues will also advance, making it possible to utilize site-directed mutagenesis to test these predictions. Future studies will employ site-directed mutagenesis to learn more about cytochrome P450 substrate access channels, to define the role of residues that do not lie within substrate recognition sites, to engineer additional soluble forms of microsomal cytochromes P450 for x-ray crystallography, and to engineer more efficient enzymes for drug activation and/or bioremediation.

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

    Science.gov (United States)

    Dods, Rachel L; Donnelly, Dan

    2015-11-23

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

  6. [Mechanism of arginine deiminase activity by site-directed mutagenesis].

    Science.gov (United States)

    Li, Lifeng; Ni, Ye; Sun, Zhihao

    2012-04-01

    Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.

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

    Science.gov (United States)

    Lee, Joanna; Daniels, Veronique; Sands, Zara A; Lebon, Florence; Shi, Jiye; Biggin, Philip C

    2015-01-01

    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. 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.

  9. Structure-function relationships of wheat flavone O-methyltransferase: Homology modeling and site-directed mutagenesis

    Directory of Open Access Journals (Sweden)

    Lim Yoongho

    2010-07-01

    Full Text Available Abstract Background Wheat (Triticum aestivum L. O-methyltransferase (TaOMT2 catalyzes the sequential methylation of the flavone, tricetin, to its 3'-methyl- (selgin, 3',5'-dimethyl- (tricin and 3',4',5'-trimethyl ether derivatives. Tricin, a potential multifunctional nutraceutical, is the major enzyme reaction product. These successive methylations raised the question as to whether they take place in one, or different active sites. We constructed a 3-D model of this protein using the crystal structure of the highly homologous Medicago sativa caffeic acid/5-hydroxyferulic acid O-methyltransferase (MsCOMT as a template with the aim of proposing a mechanism for multiple methyl transfer reactions in wheat. Results This model revealed unique structural features of TaOMT2 which permit the stepwise methylation of tricetin. Substrate binding is mediated by an extensive network of H-bonds and van der Waals interactions. Mutational analysis of structurally guided active site residues identified those involved in binding and catalysis. The partly buried tricetin active site, as well as proximity and orientation effects ensured sequential methylation of the substrate within the same pocket. Stepwise methylation of tricetin involves deprotonation of its hydroxyl groups by a His262-Asp263 pair followed by nucleophilic attack of SAM-methyl groups. We also demonstrate that Val309, which is conserved in a number of graminaceous flavone OMTs, defines the preference of TaOMT2 for tricetin as the substrate. Conclusions We propose a mechanism for the sequential methylation of tricetin, and discuss the potential application of TaOMT2 to increase the production of tricin as a nutraceutical. The single amino acid residue in TaOMT2, Val309, determines its preference for tricetin as the substrate, and may define the evolutionary differences between the two closely related proteins, COMT and flavone OMT.

  10. Homology modelling, docking, pharmacophore and site directed mutagenesis analysis to identify the critical amino acid residue of PknI from Mycobacterium tuberculosis.

    Science.gov (United States)

    Kandasamy, Srinivasan; Hassan, Sameer; Gopalaswamy, Radha; Narayanan, Sujatha

    2014-07-01

    Tuberculosis is caused by Mycobacterium tuberculosis, an intracellular pathogen. PknI is one of the 11 functional Serine/Threonine Protein Kinases which is predicted to regulate the cell division of M. tuberculosis. In order to find newer drugs and vaccine we need to understand the pathogenesis of the disease. We have used the bioinformatics approach to identify the functionally active residues of PknI and to confirm the same with wet lab experiments. In the current study, we have created homology model for PknI and have done comparative structural analysis of PknI with other kinases. Molecular docking studies were done with a library of kinase inhibitors and T95 was found as the potent inhibitor for PknI. Based on structure based pharmacophore analysis of kinase substrate complexes, Lys 41 along with Asp90, Val92 and Asp96 were identified as functionally important residues. Further, we used site directed mutagenesis technique to mutate Lys 41 to Met resulting in defective cell division of Mycobacterium smegmatis mc(2). Overall, the proposed model together with its binding features gained from pharmacophore docking studies helped in identifying ligand inhibitor specific to PknI which was confirmed by laboratory experiments. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Identification of amino acids related to catalytic function of Sulfolobus solfataricus P1 carboxylesterase by site-directed mutagenesis and molecular modeling

    Science.gov (United States)

    Choi, Yun-Ho; Lee, Ye-Na; Park, Young-Jun; Yoon, Sung-Jin; Lee, Hee-Bong

    2016-01-01

    The archaeon Sulfolobus solfataricus P1 carboxylesterase is a thermostable enzyme with a molecular mass of 33.5 kDa belonging to the mammalian hormone-sensitive lipase (HSL) family. In our previous study, we purified the enzyme and suggested the expected amino acids related to its catalysis by chemical modification and a sequence homology search. For further validating these amino acids in this study, we modified them using site-directed mutagenesis and examined the activity of the mutant enzymes using spectrophotometric analysis and then estimated by homology modeling and fluorescence analysis. As a result, it was identified that Ser151, Asp244, and His274 consist of a catalytic triad, and Gly80, Gly81, and Ala152 compose an oxyanion hole of the enzyme. In addition, it was also determined that the cysteine residues are located near the active site or at the positions inducing any conformational changes of the enzyme by their replacement with serine residues. [BMB Reports 2016; 49(6): 349-354] PMID:27222124

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

    Science.gov (United States)

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

    2005-05-15

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

  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, 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

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

    African Journals Online (AJOL)

    Construction of a high-efficiency multi-site-directed mutagenesis. ... applied to hexapeptide gene synthesis and recombinant enterokinase gene ... This method was beneficial to prepare high-quality multibase mutagenesis and also implied ...

  15. 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

    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...

  16. Site-directed mutagenesis at the human B2 receptor and molecular modelling to define the pharmacophore of non-peptide bradykinin receptor antagonists.

    Science.gov (United States)

    Meini, Stefania; Cucchi, Paola; Bellucci, Francesca; Catalani, Claudio; Faiella, Angela; Rotondaro, Luigi; Quartara, Laura; Giolitti, Alessandro; Maggi, Carlo Alberto

    2004-02-15

    Combining site-directed mutagenesis with information obtained from molecular modelling of the bradykinin (BK) human B2 receptor (hB2R) as derived from the bovine rhodopsin crystal structure [Science 289 (2000) 739], we previously defined a putative binding mode for the non-peptide B2 receptor antagonists, FR173657 and LF16-0687 [Can J Physiol Pharmacol 80 (2002) 303]. The present work is aimed to define the specific role of the quinoline moiety in the pharmacophore of these non-peptide antagonists. The effect of the mutations I110A, L114A (TM, transmembrane 3), W256A (TM6), F292A, Y295A and Y295F (TM7) was evaluated. None of the mutations affected the binding interaction of peptide ligands: the agonist BK and the peptide antagonist MEN 11270. The affinities in competing for [3H]-BK binding and in blocking the BK-induced IP production by the non-peptide antagonists LF16-0687 and FR173657 at the wild type and mutant receptors were analysed. While the affinities of LF16-0687 and FR173657 were crucially decreased at the I110A, Y295A, and Y295F mutants, the W256A mutation affected the affinity of the LF16-0687 only. The important contribution of the quinoline moiety was shown by the inability of an analogue of LF16-0687, lacking this moiety, to affect BK binding at the wild type receptor. On the other hand, the benzamidine group did not interact with mutated residues, since LF16-0687 analogues without this group or with an oxidated benzamidine displayed pairwise loss of affinity on wild type and mutated receptors. Further differences between FR173657 and LF16-0687 were highlighted at the I110 and Y295 mutants when comparing binding (pK(i)) and functional antagonist (pKB) affinity. First, the I110A mutation similarly impaired their binding affinity (250-fold), but at a less extent the antagonist potency of FR173657 only. Second, both the hydroxyl and the phenyl moieties of the Y295 residue had a specific role in the LF16-0687 interaction with the receptor, as

  17. Probing secondary glutaminyl cyclase (QC) inhibitor interactions applying an in silico-modeling/site-directed mutagenesis approach: implications for drug development.

    Science.gov (United States)

    Koch, Birgit; Buchholz, Mirko; Wermann, Michael; Heiser, Ulrich; Schilling, Stephan; Demuth, Hans-Ulrich

    2012-12-01

    Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate-modified amyloid peptides deposited in neurodegenerative disorders such as Alzheimer's disease. Inhibitors of QC are currently in development as potential therapeutics. The crystal structures of the potent inhibitor PBD150 bound to human and murine QC (hQC, mQC) have been described recently. The binding modes of a dimethoxyphenyl moiety of the inhibitor are significantly different between the structures, which contrasts with a similar K(i) value. We show the conformation of PBD150 prone to disturbance by protein-protein interactions within the crystals. Semi-empirical calculations of the enzyme-inhibitor interaction within the crystal suggest significant differences in the dissociation constants between the binding modes. To probe for interactions in solution, a site-directed mutagenesis on hQC was performed. The replacement of F325 and I303 by alanine or asparagine resulted in a 800-fold lower activity of the inhibitor, whereas the exchange of S323 by alanine or valine led to a 20-fold higher activity of PBD150. The results provide an example of deciphering the interaction mode between a target enzyme and lead substance in solution, if co-crystallization does not mirror such interactions properly. Thus, the study might provide implications for rapid screening of binding modes also for other drug targets.

  18. Improvement of megaprimer method for site-directed mutagenesis and its application to phytase

    Institute of Scientific and Technical Information of China (English)

    Haiqiang LU; Hongwei YU; Runfang GUO; Yingmin JIA

    2009-01-01

    Site-directed mutagenesis is used extensively for probing gene function. In this paper we describe an improved megaprimer method to the site-directed muta-genesis of phytase from Aspergillus niger, which allowed the mutations to be performed more efficiently in less time than other traditional methods. Three rounds of PCR and two pairs of primers were required in this method, and additionally, the restriction enzyme Dpn I was used for the elimination of template instead of the gel purification in this process. The entire procedure was performed in one tube. Moreover, this method was easier for obtaining large mutant genes than other methods. We successfully carried out the site-directed mutagenesis of phytase by adopting this method.

  19. 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.

  20. Applied an Efficient Site-directed Mutagenesis Method into Escherichia coli

    Directory of Open Access Journals (Sweden)

    Muqing Qiu

    2011-03-01

    Full Text Available A new technique for conducting site-directed mutagenesis was developed. This method allows the color selection of mutants through the simultaneous activation or deactivation of the α-peptide of ß-galactosidase. The method can efficiently create mutations at multiple sites simultaneously and can be used to perform multiple rounds of mutation on the same construct. In this paper, in order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knock-out ompR gene was constructed through overlapping PCR, digested by Notand SalⅠⅠ, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR- mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR- mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

  1. Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis

    Directory of Open Access Journals (Sweden)

    Guangrong Xie

    2016-05-01

    Full Text Available The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more “human-like” uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine–human uricase with higher homology to deduced human uricase (dHU and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1–2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7–8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H1-2P3H4P5-6H7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H1-2P3H4P5-6H7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H1-2P3H4P5-6H7-8 (E24D & E83G with increased homology (91.45% with dHU and higher activity and catalytic efficiency than the FDA-approved porcine–baboon chimera (PBC was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5–11.0 and temperature range of 20–40 °C.

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

    Science.gov (United States)

    Impellizzeri, Agata Antonina Rita; Pappalardo, Matteo; Basile, Livia; Manfra, Ornella; Andressen, Kjetil Wessel; Krobert, Kurt Allen; Messina, Angela; Levy, Finn Olav; Guccione, Salvatore

    2015-01-01

    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 (TMD) 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Improvement of PCR reaction conditions for site-directed mutagenesis of big plasmids

    Institute of Scientific and Technical Information of China (English)

    Bogdan MUNTEANU; Mario BRAUN; Kajohn BOONROD

    2012-01-01

    QuickChange mutagenesis is the method of choice for site-directed mutagenesis (SDM) of target sequences in a plasmid.It can be applied successfully to small plasmids (up to 10 kb).However,this method cannot efficiently mutate bigger plasmids.Using KOD Hot Start polymerase in combination with high performance liquid chromatography (HPLC) purified primers,we were able to achieve SDM in big plasmids (up to 16 kb) involving not only a single base change but also multiple base changes.Moreover,only six polymerase chain reaction (PCR) cycles and 0.5 μl of polymerase (instead of 18 PCR cycles and 1.0 μl of enzyme In the standard protocol) were sufficient for the reaction.

  5. Characterisation of the Rab binding properties of Rab coupling protein (RCP) by site-directed mutagenesis.

    Science.gov (United States)

    Lindsay, Andrew J; McCaffrey, Mary W

    2004-07-30

    Rab coupling protein (RCP) is a member of the Rab11-family of interacting proteins (Rab11-FIPs). Family members are characterised by their ability to interact with Rab11. This property is mediated by a conserved Rab binding domain (RBD) located at their carboxy-termini. Several Rab11-FIPs can also interact with other small GTPases. RCP interacts with Rab4 in addition to Rab11. To dissect out the individual properties of the Rab4 and Rab11 interactions with RCP, conserved amino acids within the RBD of RCP were mutated by site-directed mutagenesis. The effect of these mutations on Rab4 and Rab11 binding, and the intracellular localisation of RCP, was examined. Our results indicate that Rab11, rather than Rab4, mediates the intracellular localisation of RCP, and that the class I Rab11-FIPs compete for binding to Rab11.

  6. Improving the functional expression of a Bacillus licheniformis laccase by random and site-directed mutagenesis

    Directory of Open Access Journals (Sweden)

    Schmid Rolf D

    2009-02-01

    Full Text Available Abstract Background Laccases have huge potential for biotechnological applications due to their broad substrate spectrum and wide range of reactions they are able to catalyze. These include, for example, the formation and degradation of dimers, oligomers, polymers, and ring cleavage as well as oxidation of aromatic compounds. Potential applications of laccases include detoxification of industrial effluents, decolorization of textile dyes and the synthesis of natural products by, for instance, dimerization of phenolic acids. We have recently published a report on the cloning and characterization of a CotA Bacillus licheniformis laccase, an enzyme that catalyzes dimerization of phenolic acids. However, the broad application of this laccase is limited by its low expression level of 26 mg l-1 that was achieved in Escherichia coli. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression and activity of CotA. Results A CotA double mutant, K316N/D500G, was constructed by combining random and site-directed mutagenesis. It can be functionally expressed at an 11.4-fold higher level than the wild-type enzyme. In addition, it is able to convert ferulic acid much faster than the wild-type enzyme (21% vs. 14% and is far more efficient in decolorizing a range of industrial dyes. The investigation of the effects of the mutations K316N and D500G showed that amino acid at position 316 had a major influence on enzyme activity and position 500 had a major influence on the expression of the laccase. Conclusion The constructed double mutant K316N/D500G of the Bacillus licheniformis CotA laccase is an appropriate candidate for biotechnological applications due to its high expression level and high activity in dimerization of phenolic acids and decolorization of industrial dyes.

  7. Site-directed mutagenesis of long QT syndrome KCNQ1 gene in vitro

    Institute of Scientific and Technical Information of China (English)

    Wei LI; Junguo YANG; Rong DU; Li TIAN; Bin WANG; Qiumei XU; Qinmei KE; Qing WANG

    2008-01-01

    To construct a polymerase chain reaction (PCR) site-directed mutagenesis of the long QT syndrome KCNQ1 gene in vitro, two sets of primers were designed according to the sequence of KCNQ1 cDNA and a mismatch was introduced into primers. Mutagenesis was performed in a two-step PCR. The amplified fragments from the third PCR which contained the mutation site were sub-cloned into the T-vector pCR2.1. Then, the fragments containing the mutation site was obtained from pCR2.1 using restriction enzymes digestion and inserted into the same restriction site of plRES2-EGFP-KCNQ1. The sequencing analysis shows that the mutation site was correct. Mutation from A to G in site 983 of KCNQ1 cDNA was found. Using the Effectene transfection reagent, plRES2-EGFP-KCNQ1 (G983A) was transfected into HEK cells successfully. These results may shed light on further functional study of KCNQ1 gene.

  8. Site directed mutagenesis of StSUT1 reveals target amino acids of regulation and stability.

    Science.gov (United States)

    Krügel, Undine; Wiederhold, Elena; Pustogowa, Jelena; Hackel, Aleksandra; Grimm, Bernhard; Kühn, Christina

    2013-11-01

    Plant sucrose transporters (SUTs) are functional as sucrose-proton-cotransporters with an optimal transport activity in the acidic pH range. Recently, the pH optimum of the Solanum tuberosum sucrose transporter StSUT1 was experimentally determined to range at an unexpectedly low pH of 3 or even below. Various research groups have confirmed these surprising findings independently and in different organisms. Here we provide further experimental evidence for a pH optimum at physiological extrema. Site directed mutagenesis provides information about functional amino acids, which are highly conserved and responsible for this extraordinary increase in transport capacity under extreme pH conditions. Redox-dependent dimerization of the StSUT1 protein was described earlier. Here the ability of StSUT1 to form homodimers was demonstrated by heterologous expression in Lactococcus lactis and Xenopus leavis using Western blots, and in plants by bimolecular fluorescence complementation. Mutagenesis of highly conserved cysteine residues revealed their importance in protein stability. The accessibility of regulatory amino acid residues in the light of StSUT1's compartmentalization in membrane microdomains is discussed.

  9. Examination of the thiamin diphosphate binding site in yeast transketolase by site-directed mutagenesis.

    Science.gov (United States)

    Meshalkina, L; Nilsson, U; Wikner, C; Kostikowa, T; Schneider, G

    1997-03-01

    The role of two conserved amino acid residues in the thiamin diphosphate binding site of yeast transketolase has been analyzed by site-directed mutagenesis. Replacement of E162, which is part of a cluster of glutamic acid residues at the subunit interface, by alanine or glutamine results in mutant enzymes with most catalytic properties similar to wild-type enzyme. The two mutant enzymes show, however, significant increases in the K0.5 values for thiamin diphosphate in the absence of substrate and in the lag of the reaction progress curves. This suggests that the interaction of E162 with residue E418, and possibly E167, from the second subunit is important for formation and stabilization of the transketolase dimer. Replacement of the conserved residue D382, which is buried upon binding of thiamin diphosphate, by asparagine and alanine, results in mutant enzymes severely impaired in thiamin diphosphate binding and catalytic efficiency. The 25-80-fold increase in K0.5 for thiamin diphosphate suggests that D382 is involved in cofactor binding, probably by electrostatic compensation of the positive charge of the thiazolium ring and stabilization of a flexible loop at the active site. The decrease in catalytic activities in the D382 mutants indicates that this residue might also be important in subsequent steps in catalysis.

  10. Improvement of the Optimum Temperature of Penicillium expansum Lipase by Site-Directed Mutagenesis

    Institute of Scientific and Technical Information of China (English)

    Chen Guoren; Lin Lin

    2006-01-01

    In order to improve the optimum temperature of lipases,the Penicillum expansum lipase (PEL) gene was mutated by site-directed mutagenesis using overlap extension PCR technique.The recombinant plasmid containing mutant E83 V pPIC3.5K-lip-E83V was expressed in Pichia pastoris GS 115.Comparison experiments of the mutant PEL-E83 V-GS and the wild-type PEL-GS showed that the optimum temperature (45℃) of the mutant was 5℃ higher than that of the wild type.The thermostability of the mutant was similar to that of the wild type.The enzymatic activity of the mutant was 188 U/ml at 37℃,which was 80% that of the wild type in the same conditions.Hydrophobic interaction may be enhanced in the surface region by the hydrophilic amino acid Glu substituted with the hydrophobic amino acid Val,and may be responsible for the improvement of the optimum temperature.

  11. Activity after Site-Directed Mutagenesis of CD59 on Complement-Mediated Cytolysis

    Institute of Scientific and Technical Information of China (English)

    Xinhong Zhu; Meihua Gao; Shurong Ren; Qiubo Wang; Cunzhi Lin

    2008-01-01

    CD59 may inhibit the cytolytic activity of complement by binding to C8/C9 and protect host cell membranes against homologous membrane attack complex (MAC). However, CD59 is widely overexpressed on tumor cells,which has been implicated in tumorigenesis. The active site of CD59 relative to MAC is still confused. As reported the MAC binding site is located in the vicinity of a hydrophobic groove on the membrane distal face of the protein centered around residue W40. Here two site-directed mutagenesis were performed by overlapping extension PCR to delete residue W40 site (Mutant 1, M1) or to change C39W40K41 to W39W40W41 (Mutant 2, M2). Then we constructed mutant CD59 eukaryotic expression system and investigated their biological function on CHO cells compared with wild-type CD59. Stable populations of CHO cells expressing recombinant proteins were screened by immunotechnique. After 30 passages culturing, proteins could be tested. Dye release assays suggest that M1CD59 loses the activity against complement, while M2CD59 increases the anti-complement activity slightly.Results indicate that W40 of human CD59 is important to its activity, and prohibition of this site may be a potential way to increase complement activity and to treat tumors.

  12. [Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot].

    Science.gov (United States)

    Ke, Piyu; Huang, Jun; Hu, Sheng; Zhao, Weirui; Lü, Changjiang; Yu, Kai; Lei, Yinlin; Wang, Jinbo; Mei, Lehe

    2016-01-01

    Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity.

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

    Science.gov (United States)

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

    2002-01-01

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

  14. Viable transmembrane region mutants of bacteriophage M13 coat protein prepared by site-directed mutagenesis.

    Science.gov (United States)

    Li, Z; Deber, C M

    1991-10-31

    Bacteriophage M13 coat protein - a 50-residue protein located at the E. coli host membrane during phage reproduction - is subjected to cytoplasmic, membrane-bound, and DNA-interactive environments during the phage life cycle. In research to examine the specific features of primary/secondary structure in the effective transmembrane (TM) region of the protein (residues 21-39: YIGYAWAMVVVIVGATIGI) which modulate its capacity to respond conformationally to the progressive influences of these varying environments, we have prepared over two dozen viable mutant phages with alterations in their coat protein TM regions. Mutants were obtained through use of site-directed mutagenesis techniques in combination with three "randomized" oligonucleotides which spanned the TM region. No subcloning was required. Among mutations observed were those in which each of the four TM Val residues was changed to Ala, and several with increased Ser or Thr content, including one double Ser mutant (G23S-A25S). Polar substitutions arising at Gly23 and Tyr24-including G23D, Y24H, Y24D and Y24N-suggested that this local segment resides external to the host membrane. Milligram quantities of mutant coat proteins are obtained by growing M13 mutant phages in liter preparations, with isotopic (e.g., 13C) labelling at desired sites, for subsequent characterization and conformational analysis in membrane-mimetic media.

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

    Science.gov (United States)

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

    2015-03-01

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

  16. Site-directed mutagenesis and bacterial expression of human adenosine deaminase

    Energy Technology Data Exchange (ETDEWEB)

    Danton, M.J.; Leonardo, J.; Riley, L.; Coleman, M.S.

    1987-05-01

    Adenosine deaminase (ADA) is a purine salvage pathway enzyme, the absence of which is associated with severe combined immunodeficiency disease. Time-resolved fluorescence studies, in the presence of enzyme inhibitors, indicate that at least one of the four tryptophans present in the protein molecule is close to (or in) the active site. To investigate the role of these tryptophan residues in enzyme function, they have cloned ADA cDNA into a vector in which expression is directed by the lambda P/sub R/ promoter. E. coli cells deficient in ADA were transformed with the vector construct and were shown to synthesize catalytically active human ADA. Site directed mutagenesis, coupled with a uracil selection technique for generating mutants with high efficiency, was used to construct mutant alleles of the cloned ADA. Eight mutants were obtained with base substitutions converting each of the four tryptophans to arginine or glycine. The correlation between these specific mutations and the functional expression of ADA has been examined in the ADA deficient bacterial host.

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

    NARCIS (Netherlands)

    Mingo, J.; Erramuzpe, A.; Luna, S.; Aurtenetxe, O.; Amo, L.; Diez, I.; Schepens, J.T.G.; Hendriks, W.J.A.J.; Cortes, J.M.; Pulido, R.

    2016-01-01

    Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates compreh

  18. Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing

    Science.gov (United States)

    Fukuda, Masatora; Umeno, Hiromitsu; Nose, Kanako; Nishitarumizu, Azusa; Noguchi, Ryoma; Nakagawa, Hiroyuki

    2017-01-01

    As an alternative to DNA mutagenesis, RNA mutagenesis can potentially become a powerful gene-regulation method for fundamental research and applied life sciences. Adenosine-to-inosine (A-to-I) RNA editing alters genetic information at the transcript level and is an important biological process that is commonly conserved in metazoans. Therefore, a versatile RNA-mutagenesis method can be achieved by utilising the intracellular RNA-editing mechanism. Here, we report novel guide RNAs capable of inducing A-to-I mutations by guiding the editing enzyme, human adenosine deaminase acting on RNA (ADAR). These guide RNAs successfully introduced A-to-I mutations into the target-site, which was determined by the reprogrammable antisense region. In ADAR2-over expressing cells, site-directed RNA editing could also be performed by simply introducing the guide RNA. Our guide RNA framework provides basic insights into establishing a generally applicable RNA-mutagenesis method. PMID:28148949

  19. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol

    Directory of Open Access Journals (Sweden)

    Naismith James H

    2008-12-01

    Full Text Available Abstract Background Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable. Results We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange™ site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis. Conclusion Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange™. Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange™ but increased the overall success rates.

  20. Combination of site-directed mutagenesis and yeast surface display enhances Rhizomucor miehei lipase esterification activity in organic solvent.

    Science.gov (United States)

    Han, Shuang-yan; Zhang, Jun-hui; Han, Zhen-lin; Zheng, Sui-ping; Lin, Ying

    2011-12-01

    To increase the activity of Rhizomucor miehei lipase (RML) in organic solvent, multiple sequence alignments and rational site-directed mutagenesis were used to create RML variants. The obtained proteins were surface-displayed on Pichia pastoris by fusion to Flo1p as an anchor protein. The synthetic activity of four variants showed from 1.1- to 5-fold the activity of native lipase in an esterification reaction in heptane with alcohol and caproic acid as substrates. The increase in esterification activity may be attributed to the four mutations changing the flexibility of RML or facilitating the reaction. In conclusion, this method demonstrated that multiple sequence alignments and rational site-directed mutagenesis combined with yeast display technology is a faster and more effective means of obtaining high-efficiency esterification lipase variants compared with previous similar methods.

  1. 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

    -life of TSGAL, nine single amino acid residues were selected for site-directed mutagenesis on the basis of semi-rational design. Of these nine mutants, G305A showed half-lives of 114 min at 55 °C and 15 min at 60 °C, respectively. This is 8.6-fold higher than that of the TSGAL at 55 °C, whereas the other...

  2. Site-directed mutagenesis of bacterial cellulose synthase highlights sulfur–arene interaction as key to catalysis

    OpenAIRE

    Sun, Shi-jing; Horikawa, Yoshiki; Wada, Masahisa; SUGIYAMA, Junji; Imai, Tomoya

    2016-01-01

    Cellulose is one of the most abundant biological polymers on Earth, and is synthesized by the cellulose synthase complex in cell membranes. Although many cellulose synthase genes have been identified over the past 25 years, functional studies of cellulose synthase using recombinant proteins have rarely been conducted. In this study, we conducted a functional analysis of cellulose synthase with site-directed mutagenesis, by using recombinant cellulose synthase reconstituted in living Escherich...

  3. Site-Directed Mutagenesis of a Hyperthermophilic Endoglucanase Cel12B from Thermotoga maritima Based on Rational Design.

    Directory of Open Access Journals (Sweden)

    Jinfeng Zhang

    Full Text Available To meet the demand for the application of high activity and thermostable cellulases in the production of new-generation bioethanol from nongrain-cellulose sources, a hyperthermostable β-1,4-endoglucase Cel12B from Thermotoga maritima was selected for further modification by gene site-directed mutagenesis method in the present study, based on homology modeling and rational design. As a result, two recombinant enzymes showed significant improvement in enzyme activity by 77% and 87%, respectively, higher than the parental enzyme TmCel12B. Furthermore, the two mutants could retain 80% and 90.5% of their initial activity after incubation at 80°C for 8 h, while only 45% for 5 h to TmCel12B. The Km and Vmax of the two recombinant enzymes were 1.97±0.05 mM, 4.23±0.15 μmol·mg(-1·min(-1 of TmCel12B-E225H-K207G-D37V, and 2.97±0.12 mM, 3.15±0.21 μmol·mg(-1·min(-1 of TmCel12B-E225H-K207G, respectively, when using CMC-Na as the substrate. The roles of the mutation sites were also analyzed and evaluated in terms of electron density, hydrophobicity of the modeled protein structures. The recombinant enzymes may be used in the hydrolysis of cellulose at higher temperature in the future. It was concluded that the gene mutagenesis approach of a certain active residues may effectively improve the performance of cellulases for the industrial applications and contribute to the study the thermostable mechanism of thermophilic enzymes.

  4. Site-Directed Mutagenesis of a Hyperthermophilic Endoglucanase Cel12B from Thermotoga maritima Based on Rational Design

    Science.gov (United States)

    Zhang, Jinfeng; Shi, Hao; Xu, Linyu; Zhu, Xiaoyan; Li, Xiangqian

    2015-01-01

    To meet the demand for the application of high activity and thermostable cellulases in the production of new-generation bioethanol from nongrain-cellulose sources, a hyperthermostable β-1,4-endoglucase Cel12B from Thermotoga maritima was selected for further modification by gene site-directed mutagenesis method in the present study, based on homology modeling and rational design. As a result, two recombinant enzymes showed significant improvement in enzyme activity by 77% and 87%, respectively, higher than the parental enzyme TmCel12B. Furthermore, the two mutants could retain 80% and 90.5% of their initial activity after incubation at 80°C for 8 h, while only 45% for 5 h to TmCel12B. The Km and Vmax of the two recombinant enzymes were 1.97±0.05 mM, 4.23±0.15 μmol·mg-1·min-1 of TmCel12B-E225H-K207G-D37V, and 2.97±0.12 mM, 3.15±0.21 μmol·mg-1·min-1 of TmCel12B-E225H-K207G, respectively, when using CMC-Na as the substrate. The roles of the mutation sites were also analyzed and evaluated in terms of electron density, hydrophobicity of the modeled protein structures. The recombinant enzymes may be used in the hydrolysis of cellulose at higher temperature in the future. It was concluded that the gene mutagenesis approach of a certain active residues may effectively improve the performance of cellulases for the industrial applications and contribute to the study the thermostable mechanism of thermophilic enzymes. PMID:26218520

  5. 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.

  6. Site-directed mutagenesis of an acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti confers insecticide insensitivity.

    Science.gov (United States)

    Vaughan, A; Rocheleau, T; ffrench-Constant, R

    1997-11-01

    Insecticide resistance is a serious problem facing the effective control of insect vectors of disease. Insensitive acetylcholinesterase (AChE) confers resistance to organophosphorus (OP) and carbamate insecticides and is a widespread resistance mechanism in vector mosquitoes. Although the point mutations that underlie AChE insensitivity have been described from Drosophila, the Colorado potato beetle, and house flies, no resistance associated mutations have been documented from mosquitoes to date. We are therefore using a cloned acetylcholinesterase gene from the yellow fever mosquito Aedes aegypti as a model in which to perform site directed mutagenesis in order to understand the effects of potential resistance associated mutations. The same resistance associated amino-acid replacements as found in other insects also confer OP and carbamate resistance to the mosquito enzyme. Here we describe the levels of resistance conferred by different combinations of these mutations and the effects of these mutations on the kinetics of the AChE enzyme. Over-expression of these constructs in baculovirus will facilitate purification of each of the mutant enzymes and a more detailed analysis of their associated inhibition kinetics.

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

    Science.gov (United States)

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

    2016-01-01

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

  8. Re-evaluation of receptor-ligand interactions of the human neuropeptide Y receptor Y1: a site-directed mutagenesis study

    National Research Council Canada - National Science Library

    Sjödin, Paula; Holmberg, Sara K S; Akerberg, Helena; Berglund, Magnus M; Mohell, Nina; Larhammar, Dan

    2006-01-01

    Interactions of the human NPY (neuropeptide Y) receptor Y1 with the two endogenous agonists NPY and peptide YY and two non-peptide antagonists were investigated using site-directed mutagenesis at 17 positions...

  9. 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.

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

    Science.gov (United States)

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

    2007-07-01

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

  11. In vivo elimination of parental clones in general and site-directed mutagenesis.

    Science.gov (United States)

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

    2015-02-01

    The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5'-CCGCGG-3' and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 10(7) from a single transformation and with greater than 90% recombinant clones. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. 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

    2016-01-01

    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.

  13. Alteration of coenzyme specificity of malate dehydrogenase from Streptomyces coelicolor A3(2) by site-directed mutagenesis.

    Science.gov (United States)

    Ge, Y D; Song, P; Cao, Z Y; Wang, P; Zhu, G P

    2014-07-29

    We describe here for the first time the alteration of coenzyme specificity of malate dehydrogenase (MDH) from Streptomyces coelicolor A3(2) (ScMDH). In the present study, we replaced four amino acid residues in the Rossmann fold (βB-αC) region of NADH-dependent ScMDH by site-directed mutagenesis with those of NADPH-dependent MDH (Glu42Gly, Ile43Ser, Pro45Arg, and Ala46Ser). The coenzyme specificity of the mutant enzyme (ScMDH-T4) was examined. Coenzyme specificity of ScMDH-T4 was shifted 2231.3-fold toward NADPH using kcat/Km(coenzyme) as the measurement of coenzyme specificity. Accordingly, the effect of the replacements on coenzyme specificity is discussed. Our work provides further insight into the coenzyme specificity of ScMDH.

  14. The putative effector-binding site of Leishmania mexicana pyruvate kinase studied by site-directed mutagenesis.

    Science.gov (United States)

    Hannaert, Véronique; Yernaux, Cédric; Rigden, Daniel J; Fothergill-Gilmore, Linda A; Opperdoes, Fred R; Michels, Paul A M

    2002-03-13

    The activity of pyruvate kinase of Leishmania mexicana is allosterically regulated by fructose 2,6-bisphosphate (F-2,6-P(2)), contrary to the pyruvate kinases from other eukaryotes that are usually stimulated by fructose 1,6-bisphosphate (F-1,6-P(2)). Based on the comparison of the three-dimensional structure of Saccharomyces cerevisiae pyruvate kinase crystallized with F-1,6-P(2) present at the effector site (R-state) and the L. mexicana enzyme crystallized in the T-state, two residues (Lys453 and His480) were proposed to bind the 2-phospho group of the effector. This hypothesis was tested by site-directed mutagenesis. The allosteric activation by F-2,6-P(2) appeared to be entirely abrogated in the mutated enzymes confirming our predictions.

  15. Enhancement of thermostability and kinetic efficiency of Aspergillus niger PhyA phytase by site-directed mutagenesis.

    Science.gov (United States)

    Hesampour, Ardeshir; Siadat, Seyed Ehsan Ranaei; Malboobi, Mohammad Ali; Mohandesi, Nooshin; Arab, Seyed Shahriar; Ghahremanpour, Mohammad Mehdi

    2015-03-01

    Phytase efficiently catalyzes the hydrolysis of phytate to phosphate; it can be utilized as an animal supplement to provide animals their nutrient requirements for phosphate and to mitigate environmental pollution caused by unutilized feed phosphate. Owing to animal feed being commonly pelleted at 70 to 90 °C, phytase with a sufficiently high thermal stability is desirable. Based on the crystal structure of PhyA and bioinformatics analysis at variant heat treatments, 12 single and multiple mutants were introduced by site-directed mutagenesis in order to improve phytase thermostability. Mutated constructs were expressed in Pichia pastoris. The manipulated phytases were purified; their biochemical and kinetic investigation revealed that while the thermostability of six mutants was improved, P9 (T314S Q315R V62N) and P12 (S205N S206A T151A T314S Q315R) showed the highest heat stability (P phytase to be used as an animal feed supplement.

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

    Science.gov (United States)

    Reith, Maarten E A

    2013-07-01

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

  17. Defining HIV-1 Vif residues that interact with CBFβ by site-directed mutagenesis.

    Science.gov (United States)

    Matsui, Yusuke; Shindo, Keisuke; Nagata, Kayoko; Io, Katsuhiro; Tada, Kohei; Iwai, Fumie; Kobayashi, Masayuki; Kadowaki, Norimitsu; Harris, Reuben S; Takaori-Kondo, Akifumi

    2014-01-20

    Vif is essential for HIV-1 replication in T cells and macrophages. Vif recruits a host ubiquitin ligase complex to promote proteasomal degradation of the APOBEC3 restriction factors by poly-ubiquitination. The cellular transcription cofactor CBFβ is required for Vif function by stabilizing the Vif protein and promoting recruitment of a cellular Cullin5-RING ubiquitin ligase complex. Interaction between Vif and CBFβ is a promising therapeutic target, but little is known about the interfacial residues. We now demonstrate that Vif conserved residues E88/W89 are crucial for CBFβ binding. Substitution of E88/W89 to alanines impaired binding to CBFβ, degradation of APOBEC3, and virus infectivity in the presence of APOBEC3 in single-cycle infection. In spreading infection, NL4-3 with Vif E88A/W89A mutation replicated comparably to wild-type virus in permissive CEM-SS cells, but not in multiple APOBEC3 expressing non-permissive CEM cells. These results support a model in which HIV-1 Vif residues E88/W89 may participate in binding CBFβ.

  18. Distinct ETA receptor binding mode of macitentan as determined by site directed mutagenesis.

    Directory of Open Access Journals (Sweden)

    John Gatfield

    Full Text Available The competitive endothelin receptor antagonists (ERA bosentan and ambrisentan, which have long been approved for the treatment of pulmonary arterial hypertension, are characterized by very short (1 min occupancy half-lives at the ET(A receptor. The novel ERA macitentan, displays a 20-fold increased receptor occupancy half-life, causing insurmountable antagonism of ET-1-induced signaling in pulmonary arterial smooth muscle cells. We show here that the slow ET(A receptor dissociation rate of macitentan was shared with a set of structural analogs, whereas compounds structurally related to bosentan displayed fast dissociation kinetics. NMR analysis showed that macitentan adopts a compact structure in aqueous solution and molecular modeling suggests that this conformation tightly fits into a well-defined ET(A receptor binding pocket. In contrast the structurally different and negatively charged bosentan-type molecules only partially filled this pocket and expanded into an extended endothelin binding site. To further investigate these different ET(A receptor-antagonist interaction modes, we performed functional studies using ET(A receptor variants harboring amino acid point mutations in the presumed ERA interaction site. Three ET(A receptor residues significantly and differentially affected ERA activity: Mutation R326Q did not affect the antagonist activity of macitentan, however the potencies of bosentan and ambrisentan were significantly reduced; mutation L322A rendered macitentan less potent, whereas bosentan and ambrisentan were unaffected; mutation I355A significantly reduced bosentan potency, but not ambrisentan and macitentan potencies. This suggests that--in contrast to bosentan and ambrisentan--macitentan-ET(A receptor binding is not dependent on strong charge-charge interactions, but depends predominantly on hydrophobic interactions. This different binding mode could be the reason for macitentan's sustained target occupancy and

  19. 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.

  20. Site-directed mutagenesis in Escherichia coli of a stable R772::Ti cointegrate plasmid from Agrobacterium tumefaciens.

    Science.gov (United States)

    Hille, J; van Kan, J; Klasen, I; Schilperoort, R

    1983-01-01

    The host range of an octopine Ti plasmid is limited to Rhizobiaceae. This has been extended also to Escherichia coli in the form of a stable cointegrate with the wide-host-range plasmid R772. Its structure was studied by constructing a physical map of R772 and of the R772::pTiB6 cointegrate. An insertion sequence present in R772, called IS70, turned out to be involved in cointegrate formation. We found one intact copy of IS70 and a small segment of IS70, respectively, at the junctions of R772 and Ti DNA. The absence of a complete second copy of IS70 is a likely explanation for the stability of the cointegrate plasmid. A procedure for site-directed mutagenesis of this cointegrate plasmid in E. coli is described. The effect of mutations in the Ti plasmid part can be studied subsequently by transferring the cointegrate into Agrobacterium tumefaciens. The advantage of this procedure for Ti plasmids over other methods used at present is discussed. Images PMID:6302080

  1. Anti-oxidative effect of ribonuclease inhibitor by site-directed mutagenesis and expression in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Wu Yu; Cui Xiuyun; Wang Jihong; Zhao Peng; Xu Yuefei; Zhao Baochang

    2006-01-01

    Human placental ribonuclease inhibitor(hRI)is an acidic protein of Mr-50kDa with unusually high contents of leucine and cysteine residues.It is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonuclease.hRI has 32 cysteine residues,and the oxidative formation of disulfide bonds from those cysteine residues is a rapid cooperative process that inactivates hRI.The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence.In the present aork,two molecules of alanine substituting for Cys328 and Cys329 were performed by site-directed mutagenesis.The site-mutated RI cDNA was constructed into plasmid pPIC9K and then transformed Pichia pastoris GS115 by electroporation.After colony screening,the bacterium was cultured and the product Was purified with affinity chromatography.The affinity of the recombinant human RI with double site mutation was examined for RNase A and its anti-oxidative effect.Results indicated that there were not many changes in the affinity for RNase A detected when compared with the wild type of RI.But the capacity of anti-oxidative effect increased by 7~9 times.The enhancement in anti-oxidative efrect might be attributed to preventing the formation of disulfide bond between Cys328 and Cys329 and the three dimensional structure of RI was thereby maintained.

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

    Directory of Open Access Journals (Sweden)

    Silvia Schumann

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

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

    Directory of Open Access Journals (Sweden)

    Farnaz Nikzad Jamnani

    2013-11-01

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

  4. 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.

  5. 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.

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

    Science.gov (United States)

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

    2010-12-10

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

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

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    Yates Robert B

    2002-02-01

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

  8. Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site.

    Science.gov (United States)

    Prado, R A; Barbosa, J A; Ohmiya, Y; Viviani, V R

    2011-07-01

    The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases.

  9. Modulation of the reactivity of multiheme cytochromes by site-directed mutagenesis: moving towards the optimization of microbial electrochemical technologies.

    Science.gov (United States)

    Alves, Alexandra S; Costa, Nazua L; Tien, Ming; Louro, Ricardo O; Paquete, Catarina M

    2017-01-01

    Dissimilatory metal-reducing bacteria perform extracellular electron transfer, a metabolic trait that is at the core of a wide range of biotechnological applications. To better understand how these microorganisms transfer electrons from their metabolism to an extracellular electron acceptor, it is necessary to characterize in detail the key players in this process, the multiheme c-type cytochromes. Shewanella oneidensis MR-1 is a model organism for studying extracellular electron transfer, where the heme protein referred to as small tetraheme cytochrome is one of the most abundant multiheme cytochromes found in the periplasmic space of this bacterium. The small tetraheme cytochrome is responsible for the delivery of electrons to the porin-cytochrome supercomplexes that permeate the outer-membrane and reduce metallic minerals or electrodes. In this work, well-established thermodynamic and kinetic models that discriminate the electron transfer activity of the four individual hemes were employed to characterize a set of single amino-acid mutants of the small tetraheme cytochrome and their interaction with small inorganic electron donors and acceptors. The results show that electrostatics play an important role in the reactivity of the small tetraheme cytochrome with small inorganic electron partners, in particularly in the kinetics of the electron transfer processes. This thorough exploration using site-directed mutants provides key mechanistic insights to guide the rational manipulation of the proteins that are key players in extracellular electron transfer processes, towards the improvement of microbial electrochemical applications using dissimilatory metal-reducing bacteria.

  10. [Effects of the site-directed mutagenesis at nsP2-726Pro on replicon vector derived from XJ-160 virus].

    Science.gov (United States)

    Tang, Li; Zhu, Wu-Yang; Fu, Shi-Hong; He, Ying; Wang, Zhi-Yu; Liang, Guo-Dong

    2010-03-01

    To investigate the effects of site-directed mutagenesis at nsP2-726Pro on the characteristics of replicon vector derived from XJ-160 virus, a Sindbis virus (SINV) isolated in China. The mutant vector pBRep-726L, pBRep-726S, pBRep-726V or pBRep-726A was constructed by introducing nsP2-726Pro --> Leu, nsP2-726Pro --> Ser, nsP2-726Pro --> Val or nsP2-726Pro --> Ala into XJ-160 viral replicon vector pBRepXJ respectively. To quantitatively and qualitatively determine the site-directed mutagenesis on the replicon, the recombinant plasmids expressing Neomycinr (Neo(r)), enhanced green fluorescent protein (EGFP) or Renilla luciferase (R. luc) were constructed by cloning report genes into pBRepXJ or mutant XJ-160 vector respectively. And in vitro-synthesized RNA from expression vectors were electroporated into BHK-21 cells. Compared with the wild-type replicon, the mutation nsP2-726Pro --> Val or nsP2-726Pro --> Ala accelerated the processing of CPE on BHK-21 cells and simultaneously enhanced its self-replicating capacity. The mutant vector pBRep-726L with Leu substitution exhibited similar packaging capacity to that of pBRepXJ. In contrast, pBRep-726S exhibited a medium phenotype, including the process of CPE and the activity of R. luc expression in BHK-21 cells. The site-directed mutagenesis at nsP2-726Pro not only regulates directly XJ-160 virus vector-host cell interactions, but also plays an important role in its packaging capacity. All of these results lay a basis for researching the relation between the structure and function of alphavirus genome and developing alphavirus vector system with Chinese intellectual property.

  11. 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…

  12. Site-directed mutagenesis of the toxin from the Chinese scorpion Buthus martensii Karsch (BmKAS): insight into sites related to analgesic activity.

    Science.gov (United States)

    Cui, Yong; Song, Yong-Bo; Ma, Lin; Liu, Yan-Feng; Li, Guo-Dong; Wu, Chun-Fu; Zhang, Jing-Hai

    2010-10-01

    This study utilized the E. coli expression system to investigate the role of amino acid residues in toxin from the Chinese scorpion--Buthus martensii Karsch (BmKAS). To evaluate the extent to which residues of the toxin core contribute to its analgesic activity, ten mutants of BmKAS were obtained by PCR. Using site-directed mutagenesis, all of these residues were substituted with different amino acids. This study represents a thorough mapping and elucidation of the epitopes that form the molecular basis of the toxin's analgesic activity. Our results showed large mutant-dependent differences that emphasize the important roles of the studied residues.

  13. Serine-202 is the putative precursor of the active site dehydroalanine of phenylalanine ammonia lyase. Site-directed mutagenesis studies on the enzyme from parsley (Petroselinum crispum L.).

    Science.gov (United States)

    Schuster, B; Rétey, J

    1994-08-01

    To investigate the possible role of serine as a precursor of dehydroalanine at the active site of phenylalanine ammonia lyase, two serines, conserved in all known PAL and histidase sequences, were changed to alanine by site-directed mutagenesis. The resulting mutant genes were subcloned into the expression vector pT7.7 and the gene products were assayed for PAL activity. Mutant PALMutS209A showed the same catalytic property as wild-type PAL, whereas mutant PALMutS202A was devoid of catalytic activity, indicating that serine-202 is the most likely precursor of the active site dehydroalanine.

  14. Activity modulation of the oligopeptidase B from Serratia proteamaculans by site-directed mutagenesis of amino acid residues surrounding catalytic triad histidine.

    Science.gov (United States)

    Mikhailova, Anna G; Rakitina, Tatiana V; Timofeev, Vladimir I; Karlinsky, David M; Korzhenevskiy, Dmitry A; Agapova, Yulia К; Vlaskina, Anna V; Ovchinnikova, Marina V; Gorlenko, Valentina A; Rumsh, Lev D

    2017-08-01

    Oligopeptidase B (OpdB; EC 3.4.21.83) is a trypsin-like peptidase belonging to the family of serine prolyl oligopeptidases; two-domain structure of the enzyme includes C-terminal peptidase catalytic domain and N-terminal seven-bladed β-propeller domain. Importance of the interface between these domains and particularly of the 5 salt bridges for enzyme activity was established for protozoan OpdBs. However, these salt bridges are not conserved in γ -proteobacterial OpdBs including the peptidase from Serratia proteamaculans (PSP). In this work, using comparative modelling and protozoan OpdBs' crystal structures we created 3D models of PSP in open and closed forms to elucidate the mechanism underlying inactivation of the truncated form of PSP1-655 obtained earlier. Analysis of the models shows that in the closed form of PSP charged amino acid residues of histidine loop, surrounding the catalytic triad His652, participate in formation of the inter-domain contact interface between catalytic and β-propeller domains, while in the open form of PSP disconnection of the catalytic triad and distortion of these contacts can be observed. Complete destruction of this interface by site-directed mutagenesis causes inactivation of PSP while elimination of the individual contacts leads to differential effects on the enzyme activity and substrate specificity. Thus, we identified structural factors regulating activity of PSP and supposedly of other γ-proteobacterial OpdBs and discovered the possibility of directed modulation of their enzymatic features. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. Modulation of replication efficacy of the hepatitis C virus replicon Con1 by site-directed mutagenesis of an NS4B aminoterminal basic leucine zipper.

    Science.gov (United States)

    Welker, M-W; Susser, S; Welsch, C; Perner, D; Füller, C; Kronenberger, B; Herrmann, E; Zeuzem, S; Sarrazin, C

    2012-11-01

    The hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is assumed to function as a membrane anchor and protein hub for the viral replication complex. The aim of the current work was to modulate HCV replication efficacy in the subgenomic Con1 replicon by mutations of specific sites within the aminoterminal-located basic leucine zipper (bZIP), a candidate motif for protein-protein interactions involving NS4B. Mutational sites and amino acid substitutes were determined by in-silico sequence analyses of the NS4B-bZIP motif in 357 isolates of HCV genotype 1b from the euHCVdB and LosAlamos database and consecutive analysis of conserved physico-chemical properties at bZIP specific positions. Mutants with predicted minor, medium or major reduction of replication efficacy were tested in the pFKI389neo/NS3-3'/ET plasmid replicon model. Four sites (L25, T29, V39 and W43) of crucial importance for bZIP-mediated protein interaction with predicted apolarity of respective amino acid positions were selected for mutational studies. Substitutes with physico-chemical properties matching the predicted requirements either well (T29A), moderately (L25W, V39W), or insufficiently (T29E, W43E) were associated with slightly improved, moderate and marked decreased replication efficacy, respectively. Spontaneous (T29G) and adaptive (A28G, E40G) mutations occurred in the T29E mutation isolate only and were associated with marked reduction of replication efficacy. The bZIP motif region of NS4B is crucial for RNA replication in the subgenomic Con1 replicon system. RNA replication efficacy can be modulated by site-directed mutagenesis at specific bZIP functional sites. New adaptive amino acid mutations were identified within the HCV NS4B protein.

  16. 阿维链霉菌aveD基因的定点突变%Site-directed Mutagenesis of Streptomyces avermitilis aveD Gene

    Institute of Scientific and Technical Information of China (English)

    汤晖; 张利平

    2011-01-01

    [Objective] The aim of this study was to produce Streptomyces avermitilis strain with site-directed mutagenesis in aveD gene,and so as to provide theoretical basis for genetic breeding of S.avermitilis.[Method] PCR-driven overlap extension was conducted for the site-directed mutagenesis in aveD gene;the mutated aveD gene then was used to construct vector pDC3(pKC1139∷aveD) via molecular manipulations like in vitro enzyme digestion and ligation;the vector pDC3(pKC1139∷aveD) was then introduced to aveD deletion mutant 489 of avermectin-producing strain S.avermitilis 76-9.[Result] Mutant strain 536 of site-directed mutagenesis of S.avermitilis 76-9 was obtained by homologous recombination.The sequencing results show that the sixty-ninth base C in aveD-coding region of mutant 536 was substituted by T,and the corresponding amino acid Thr was mutated to be Ile.[Conclusion] This study laid basis for the development of strains specifically producing avermectin B.%[目的]获得aveD基因定点突变株,为阿维菌素的遗传育种提供理论依据。[方法]采用重叠延伸PCR技术对aveD基因进行定点突变,并通过体外酶切连接等分子生物学操作,构建了aveD基因的定点突变载体pDC3(pKC1139∷aveD、),导入阿维菌素(Avermectin)产生菌阿维链霉菌(Streptomyces avermitilis)76-9的aveD基因缺失突变株489中。[结果]经过同源重组,获得aveD基因定点缺失突变株536。测序结果表明突变株536,aveD基因编码区中第69位碱基C突变为T,相应的氨基酸序列第23位由Thr突变为Ile。[结论]该研究为研制生产阿维菌素B的基因工程菌奠定了基础。

  17. A saxitoxin-binding aptamer with higher affinity and inhibitory activity optimized by rational site-directed mutagenesis and truncation.

    Science.gov (United States)

    Zheng, X; Hu, B; Gao, S X; Liu, D J; Sun, M J; Jiao, B H; Wang, L H

    2015-07-01

    Saxitoxin (STX), a member of the family of paralytic shellfish poisoning toxins, poses toxicological and ecotoxicological risks. To develop an analytical recognition element for STX, a DNA aptamer (APT(STX1)) was previously discovered via an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by Handy et al. Our study focused on generating an improved aptamer based on APT(STX1) through rational site-directed mutation and truncation. In this study, we generated the aptamer, M-30f, with a 30-fold higher affinity for STX compared with APT(STX1). The Kd value for M-30f was 133 nM, which was calculated by Bio-Layer Interferometry. After optimization, we detected and compared the interaction of STX with aptamers (APT(STX1) or M-30f) through several techniques (ELISA, cell bioassay, and mouse bioassay). Both aptamers' STX-binding ability was demonstrated in all three methods. Moreover, M-30f performs better than its parent sequence with higher suppressive activity against STX. As a molecular recognition element, M-30f has good prospects for practical application. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme.

    Science.gov (United States)

    Malcolm, B A; Rosenberg, S; Corey, M J; Allen, J S; de Baetselier, A; Kirsch, J F

    1989-01-01

    The roles of the catalytic active-site residues aspartic acid-52 and glutamic acid-35 of chicken lysozyme (EC 3.2.1.17) have been investigated by separate in vitro mutagenesis of each residue to its corresponding amide (denoted as D52N and E35Q, respectively). The mutant enzyme D52N exhibits approximately 5% of the wild-type lytic activity against Micrococcus luteus cell walls, while there is no measurable activity associated with E35Q (0.1% +/- 0.1%). The measured dissociation constants for the chitotriose-enzyme complexes were 4.1 microM (D52N) and 13.4 microM (E35Q) vs. 8.6 microM for wild type, indicating that the alterations in catalytic properties may be due in part to binding effects as well as to direct catalytic participation of these residues. The mutant lysozymes have been expressed in and secreted from yeast and obtained at a level of approximately 5 mg per liter of culture by high-salt elution from the cell walls.

  19. 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

    thereof on the kinetics of uPA binding in real-time by surface plasmon resonance. Only four positions in loop 3 of uPAR domain I exhibited significant changes in the contribution to the free energy of uPA binding (DeltaDeltaG >/= 1.3 kcal mol(-1)) upon single-site substitutions to alanine (i.e. Arg(53...... the possible involvement of uPAR domain I and particularly loop 3 thereof in ligand binding (Ploug, M. (1998) Biochemistry 37, 16494-16505). Guided by these results we have now performed an alanine scanning analysis of this region in uPAR by site-directed mutagenesis and subsequently measured the effects......), 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...

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

    Science.gov (United States)

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

    1996-11-01

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

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

    Science.gov (United States)

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

    2015-05-01

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

  2. Mapping the lipoylation site of Arabidopsis thaliana plastidial dihydrolipoamide S-acetyltransferase using mass spectrometry and site-directed mutagenesis.

    Science.gov (United States)

    Casteel, Jill; Miernyk, Ján A; Thelen, Jay J

    2011-11-01

    Catalytic enhancement achieved by the pyruvate dehydrogenase complex (PDC) results from a combination of substrate channeling plus active-site coupling. The mechanism for active-site coupling involves lipoic acid prosthetic groups covalently attached to Lys in the primary sequence of the dihydrolipoyl S-acetyltransferase (E2) component. Arabidopsis thaliana plastidial E2 (AtplE2-1A-His(6)) was expressed in Escherichia coli. Analysis of recombinant protein by SDS-PAGE revealed a Mr 59,000 band. Supplementation of bacterial culture medium with l-lipoic acid (LA) shifted the band to Mr 57,000. Intact mass determinations using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) revealed the faster migrating E2 species was 189 Da larger than the slower migrating form, exactly the difference that would result from addition of a single lipoamide group. Results from systematic MALDI-TOF analysis of Lys-containing tryptic peptides derived from purified recombinant AtplE2-1A indicate that Lys96 is the site of lipoyl-addition. Analysis of Lys96 site-directed mutant proteins showed that they migrated as single species during SDS-PAGE when expressed in either the absence or presence of supplemental LA. Results from both intact and tryptic peptide mass determinations by MALDI-TOF MS confirmed that the mutant proteins were not lipoylated. The A. thaliana plastidial E2 subunit includes a single lipoyl-prosthetic group covalently attached to Lys96. Despite low primary sequence identity with bacterial E2, the plant E2 protein was recognized and modified by E. coli E2 lipoyl-addition system. Results from meta-genomic analysis suggest a β-turn is more important in defining the site for LA addition than a conserved sequence motif.

  3. Purification and site-directed mutagenesis of linoleate 9S-dioxygenase-allene oxide synthase of Fusarium oxysporum confirms the oxygenation mechanism.

    Science.gov (United States)

    Chen, Yang; Jernerén, Fredrik; Oliw, Ernst H

    2017-07-01

    Plants and fungi form jasmonic acid from α-linolenic acid. The first two steps of biosynthesis in plants occur by sequential transformation by 13S-lipoxygenase and allene oxide synthase (AOS). The biosynthesis in fungi may follow this classical scheme, but the only fungal AOS discovered so far are cytochromes P450 (CYP) fused to 8- and 9-dioxygenases (DOX). In the present report, we purified recombinant 9S-DOX-AOS of Fusarium oxysporum from cell lysate by cobalt affinity chromatography to near homogeneity and studied key residues by site-directed mutagenesis. Sequence homology with 8R-DOX-linoleate diol synthases (8R-DOX-LDS) suggested that Tyr414 catalyzes hydrogen abstraction and that Cys1051 forms the heme thiolate ligand. Site-directed mutagenesis (Tyr414Phe; Cys1051Ser) led to loss of 9S-DOX and 9S-AOS activities, respectively, but other important residues in the CYP parts of 5,8- and 7,8-LDS or 9R-AOS were not conserved. The UV-visible spectrum of 9S-DOX-AOS showed a Soret band at 409 nm, which shifted to 413 nm in the Cys1051Ser mutant. The 9S-AOS of the Tyr414Phe mutant transformed 9S-hydroperoxides of α-linolenic and linoleic acids to allene oxides/α-ketols, but it did not transform 13-hydroperoxides. We conclude that 9S- and 8R-DOX catalyze hydrogen abstraction at C-11 and C-8, respectively, by homologous Tyr residues. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Probing the aromatic-donor-binding site of horseradish peroxidase using site-directed mutagenesis and the suicide substrate phenylhydrazine.

    Science.gov (United States)

    Gilfoyle, D J; Rodriguez-Lopez, J N; Smith, A T

    1996-03-01

    The haem groups from two classes of site-directed mutants of horseradish peroxidase isoenzyme C (HRP-C) (distal haem pocket mutants, [H42L]HRP-C* and [R38K]-HRP-C* and peripheral-haem-access-channel mutants, [F142A]HRP-C* and [F143A]HRP-C*) were extracted and analysed by reverse-phase HPLC after phenylhydrazine-induced suicide inactivation. The relative abundance of the two covalently modified haems, C20-phenyl (delta-meso phenyl) and C18-hydroxymethyl haem, provided a sensitive topological probe for changes induced in the protein architecture in the vicinity of the haem active site and substrate-access channel. Although differing considerably in their efficiency as peroxidases ([H42L]HRP-C* exhibited only approximately 0.03% of the peroxidase activity of wild type), the variants studied gave rise to a modification pattern typical of an exposed haem edge thereby strengthening the argument that it is the overall protein topology rather than the intrinsic catalytic activity of the active site that determines the sites of covalent haem modification. Mutants which showed impaired ability to bind the aromatic donor benzhydroxamic acid were less readily modified by the phenyl radical at the haem C18-methyl position although the level of arylation at the haem C20 position remained remarkable constant. Our findings suggest that the overall efficacy of haem modification catalysed by HRP-C during turnover with phenylhydrazine and its vulnerability towards inactivation are related to its general ability to bind aromatic donor molecules. Results from phenylhydrazine treatment of HRP-C wild-type and mutant variants were compared with those obtained for Coprinus cinereus peroxidase, an enzyme which from its structure is known to have a remarkably open access channel to the haem edge. We show evidence that C. cinereus peroxidase is able to bind benzhydroxamic acid, albeit with a relatively high Kd (Kd 3.7 mM), a probe for aromatic-donor binding. We suggest reasons why

  5. Site-directed mutagenesis of Arginine282 suggests how protons and peptides are co-transported by rabbit PepT1.

    Science.gov (United States)

    Pieri, Myrtani; Hall, Dashiell; Price, Richard; Bailey, Patrick; Meredith, David

    2008-01-01

    The mammalian proton-coupled peptide transporter PepT1 is the major route of uptake for dietary nitrogen, as well as the oral absorption of a number of drugs, including beta-lactam antibiotics and angiotensin-converting enzyme inhibitors. Here we have used site-directed mutagenesis to investigate further the role of conserved charged residues in transmembrane domains. Mutation of rabbit PepT1 arginine282 (R282, transmembrane domain 7) to a positive (R282K) or physiologically titratable residue (R282H), resulted in a transporter with wild-type characteristics when expressed in Xenopus laevis oocytes. Neutral (R282A, R282Q) or negatively charged (R282D, R282E) substitutions gave a transporter that was not stimulated by external acidification (reducing pH(out) from 7.4 to 5.5) but transported at the same rate as the wild-type maximal rate (pH(out) 5.5); however, only the R282E mutation was unable to concentrate substrate above the extracellular level. All of the R282 mutants showed trans-stimulation of efflux comparable to the wild-type, except R282E-PepT1 which was faster. A conserved negatively charged residue, aspartate341 (D341) in transmembrane domain 8 was implicated in forming a charge pair with R282, as R282E/D341R- and R282D/D341R-PepT1 had wild-type transporter characteristics. Despite their differences in ability to accumulate substrate, both R282E- and R282D-PepT1 showed an increased charge:peptide stoichiometry over the wild-type 1:1 ratio for the neutral dipeptide Gly-l-Gln, measured using two-electrode voltage clamp. This extra charge movement was linked to substrate transport, as 4-aminobenzoic acid, which binds but is not translocated, did not induce membrane potential depolarisation in R282E-expressing oocytes. A model is proposed for the substrate binding/translocation process in PepT1.

  6. Engineering of cytochrome P450 3A4 for enhanced peroxide-mediated substrate oxidation using directed evolution and site-directed mutagenesis.

    Science.gov (United States)

    Kumar, Santosh; Liu, Hong; Halpert, James R

    2006-12-01

    CYP3A4 has been subjected to random and site-directed mutagenesis to enhance peroxide-supported metabolism of several substrates. Initially, a high-throughput screening method using whole cell suspensions was developed for H2O2-supported oxidation of 7-benzyloxyquinoline. Random mutagenesis by error-prone polymerase chain reaction and activity screening yielded several CYP3A4 mutants with enhanced activity. L216W and F228I showed a 3-fold decrease in Km, HOOH and a 2.5-fold increase in kcat/Km, HOOH compared with CYP3A4. Subsequently, T309V and T309A were created based on the observation that T309V in CYP2D6 has enhanced cumene hydroperoxide (CuOOH)-supported activity. T309V and T309A showed a > 6- and 5-fold higher kcat/Km, CuOOH than CYP3A4, respectively. Interestingly, L216W and F228I also exhibited, respectively, a > 4- and a > 3-fold higher kcat/Km, CuOOH than CYP3A4. Therefore, several multiple mutants were constructed from rationally designed and randomly isolated mutants; among them, F228I/T309A showed an 11-fold higher kcat/Km, CuOOH than CYP3A4. Addition of cytochrome b5, which is known to stimulate peroxide-supported activity, enhanced the kcat/Km, CuOOH of CYP3A4 by 4- to 7-fold. When the mutants were tested with other substrates, T309V and T433S showed enhanced kcat/Km, CuOOH with 7-benzyloxy-4-(trifluoromethyl)coumarin and testosterone, respectively, compared with CYP3A4. In addition, in the presence of cytochrome b5, T433S has the potential to produce milligram quantities of 6beta-hydroxytestosterone through peroxide-supported oxidation. In conclusion, a combination of random and site-directed mutagenesis approaches yielded CYP3A4 enzymes with enhanced peroxide-supported metabolism of several substrates.

  7. 基于柑橘绿霉菌CYP51同源模建的定点突变与抗性机制分析%Site-directed mutagenesis and resistant analysis based on the homology modeling of Penicillium digitatum CYP51

    Institute of Scientific and Technical Information of China (English)

    张建华; 熊丽; 夏诗慧; 王金龙; 李倩; 刘德立

    2012-01-01

    Penicillium digitatum sterol 14α methylation enzyme(PdCYP51),the targeted enzyme of azole fungicides,was widely applied in preventing the disease caused by P.digitatum.To elucidate the mechanism of interaction between the PdCYP51 and fungicides,3D-structure of PdCYP51 was built based on the eukaryotes human CYP51 crystal structure,and commercial diniconazole was docked into the active cavity of PdCYP51.The key amino acids which influence the interaction between the PdCYP51 and diniconazole were predicted,and the mutants(PdCYP51-Y112H,F120L,F120D and S309A) were obtained by site-directed mutagenesis.The results indicated that the expression of mutants changed differently and PdCYP51-Y112H and F120L mutants keep the same amount with PdCYP51;while PdCYP51-F120D and S309A increased.Compared with the wide PdCYP51(0.12 μmol/L),the affinity Kd values of these four mutants against fungicide determined by spectral analysis increased,with 1.28,0.18,1.03,1.31 μmol/L,respectively,and their binding activity with diniconazole reduced.These observations suggest that three sites are key amino acids,and hydrophobic cavity and stable force is the important factors in binding between PdCYP51 and diniconazole.%为阐明杀菌剂与PdCYP51的相互作用及其抗性机制,基于最新解析的真核生物人类的CYP51晶体结构,同源模建了PdCYP51的三维结构,并选取商品化的杀菌剂烯唑醇进行分子对接,预测影响PdCYP51与烯唑醇相互作用的关键氨基酸。采用定点突变技术获得了PdCYP51-Y112H、F120L、F120D和S309A 4种突变体(PdCYP51m)。结果表明,突变体蛋白PdCYP51-Y112H、F120L、F120D和S309A表达量均有不同程度的变化,PdCYP51-Y112H和F120L与未突变蛋白表达量相当,而PdCYP51-F120D和S309A表达量增加。4种突变体与杀菌剂的结合能力降低,其结合常数分别为1.28、0.18、1.03和1.31μmol/L,均大于未突变PdCYP51的0.12μmol/L。表明这些氨基酸是

  8. Identification of ω-aminotransferase from Caulobacter crescentus and site-directed mutagenesis to broaden substrate specificity.

    Science.gov (United States)

    Hwang, Bum-Yeol; Ko, Seung-Hyun; Park, Hyung-Yeon; Seo, Joo-Hyun; Lee, Bon-Su; Kim, Byung-Gee

    2008-01-01

    A putative aminotransferase gene, cc3143 (aptA), from Caulobacter crescentus was screened by bioinformatical tools and overexpressed in E. coli, and the substrate specificity of the aminotransferase was investigated. AptA showed high activity for short-chain beta-amino acids. It showed the highest activity for 3-amino-n-butyric acid. It showed higher activity toward aromatic amines than aliphatic amines. The 3D model of the aminotransferase was constructed by homology modeling using a dialkylglycine decarboxylase PDB ID: 1DGE) as a template. Then, the aminotransferase was rationally redesigned to increase the activity for 3-amino- 3-phenylpropionic acid. The mutants N285A and V227G increased the relative activity for 3-amino-3-phenylpropionic acid to 3-amino-n-butyric acid by 11-fold and 3-fold, respectively, over that of wild type.

  9. 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.

  10. Probing of the location of the allosteric site on m1 muscarinic receptors by site-directed mutagenesis.

    Science.gov (United States)

    Matsui, H; Lazareno, S; Birdsall, N J

    1995-01-01

    In an attempt to locate the allosteric site on muscarinic receptors to which gallamine binds, 21 residues in the putative external loops and loop/transmembrane helix interfaces have been mutated to alanine. These residues are conserved in mammalian m1-m5 receptors. All mutant receptors can be expressed in COS-7 cells at high levels and appear to be functional, in that acetylcholine binding is sensitive to GTP. The gallamine binding site does not appear to involve the first, second, and most of the third extracellular loops. Tryptophan-400 and -101 inhibit gallamine binding when mutated to alanine or to phenylalanine and may form part of the allosteric site. Several mutations also affect antagonist binding. Surprisingly, tryptophan-91, a residue conserved in monoamine and peptide receptors, is important for antagonist binding. This residue, present in the middle of the first extracellular loop, may have a structural role in many G protein-coupled receptors. Antagonist binding is also affected by mutations of tryptophan-101 and tyrosine-404 to alanine or phenylalanine. In a helical wheel model, trytophan-101 and tyrosine-404, in conjunction with serine-78, aspartate-105, and tyrosine-408, form a cluster of residues that have been reported to affect antagonist binding when mutated, and they may therefore be part of the antagonist binding site. It is suggested that the allosteric site may be located close to and just extracellular to the antagonist binding site. The binding of methoctramine, an antagonist with allosteric properties, is not substantially affected by mutations at tryptophan-91, -101, and -400 and tyrosine-404, and thus these amino acids are not important for its binding. The binding of himbacine, another antagonist with allosteric properties, is affected by these mutations but in a manner different from that of gallamine or competitive antagonists. It has not been possible to determine whether methoctramine and himbacine bind exclusively to the

  11. Hypericum perforatum hydroxyalkylpyrone synthase involved in sporopollenin biosynthesis--phylogeny, site-directed mutagenesis, and expression in nonanther tissues.

    Science.gov (United States)

    Jepson, Christina; Karppinen, Katja; Daku, Rhys M; Sterenberg, Brian T; Suh, Dae-Yeon

    2014-09-01

    Anther-specific chalcone synthase-like enzyme (ASCL), an ancient plant type III polyketide synthase, is involved in the biosynthesis of sporopollenin, the stable biopolymer found in the exine layer of the wall of a spore or pollen grain. The gene encoding polyketide synthase 1 from Hypericum perforatum (HpPKS1) was previously shown to be expressed mainly in young flower buds, but also in leaves and other tissues at lower levels. Angiosperm ASCLs, identified by sequence and phylogenetic analyses, are divided into two sister clades, the Ala-clade and the Val-clade, and HpPKS1 belongs to the Ala-clade. Recombinant HpPKS1 produced triketide and, to a lesser extent, tetraketide alkylpyrones from medium-chain (C6) to very long-chain (C24) fatty acyl-CoA substrates. Like other ASCLs, HpPKS1 also preferred hydroxyl fatty acyl-CoA esters over the analogous unsubstituted fatty acyl-CoA esters. To study the structural basis of the substrate preference, mutants of Ala200 and Ala215 at the putative active site and Arg202 and Asp211 at the modeled acyl-binding tunnel were constructed. The A200T/A215Q mutant accepted decanoyl-CoA, a poor substrate for the wild-type enzyme, possibly because of active site constriction by bulkier substitutions. The substrate preference of the A215V and A200T/A215Q mutants shifted toward nonhydroxylated, medium-chain to long-chain fatty acyl-CoA substrates. The R202L/D211V double mutant was selective for acyl-CoA with chain lengths of C16-C18, and showed a diminished preference for the hydroxylated acyl-CoA substrates. Transient upregulation by abscisic acid and downregulation by jasmonic acid and wounding suggested that HpPKS1, and possibly other Ala-clade ASCLs, may be involved in the biosynthesis of minor cell wall components in nonanther tissues.

  12. In vitro models of mutagenesis.

    Science.gov (United States)

    Strauss, B S; Larson, K; Sagher, D; Rabkin, S; Shenkar, R; Sahm, J

    1985-01-01

    The bypass of lesions in DNA with insertion of nucleotides opposite damaged bases has been studied as a model for mutagenesis in an in vitro system. Lesions introduced by dimethyl sulfate at adenines and by ultraviolet light at pyrimidine dimers act as termination sites on both double- and single-stranded DNA templates. Base selection opposite noninformational lesions is, in part, a property of the polymerases: different polymerases have different selectivities although all polymerases tested seem to prefer purines. The ability to insert "incorrect" bases is determined in part by the sequence 5' to the lesion on the template strand. The hypothesis that damaged purines tend to result in transversions can be applied to published data on activation of the c-ras oncogene.

  13. Improvement of the optimum pH of Aspergillus niger xylanase towards an alkaline pH by site-directed mutagenesis.

    Science.gov (United States)

    Li, Fei; Xie, Jingcong; Zhang, Xuesong; Zhao, Linguo

    2015-01-01

    In an attempt to shift the optimal pH of the xylanase B (XynB) from Aspergillus niger towards alkalinity, target mutation sites were selected by alignment between Aspergillus niger xylanase B and other xylanases that have alkalophilic pH optima that highlight charged residues in the eight-residues-longer loop in the alkalophilic xylanase. Multiple engineered XynB mutants were created by site-directed mutagenesis with substitutions Q164K and Q164K+D117N. The variant XynB-117 had the highest optimum pH (at 5.5), which corresponded to a basic 0.5 pH unit shift when compared with the wild-type enzyme. However, the optimal pH of the XynB- 164 mutation was not changed, similar to the wild type. These results suggest that the residues at positions 164 and 117 in the eight-residues-longer loop and the cleft's edge are important in determining the pH optima of XynB from Aspergillus niger.

  14. Improvement in thermostability of metagenomic GH11 endoxylanase (Mxyl) by site-directed mutagenesis and its applicability in paper pulp bleaching process.

    Science.gov (United States)

    Satyanarayana, Digvijay Verma T

    2013-12-01

    An attempt has been made for enhancing the thermostability of xylanase (Mxyl) retrieved from a compost-soil-based metagenomic library. The analysis of the structure of xylanase by molecular dynamics simulation revealed more structural fluctuations in β-sheets. When the surface of β-sheets was enriched with arginine residues by substituting serine/threonine by site-directed mutagenesis, the enzyme with four arginine substitutions (MxylM4) exhibited enhanced thermostability at 80 °C. The T 1/2 of MxylM4 at 80 °C, in the presence of birchwood xylan, increased from 130 to 150 min at 80 °C without any alteration in optimum pH and temperature and molecular mass. Improvement in thermostability of MxylM4 was corroborated by increase in T m by 6 °C over that of Mxyl. The K m of MxylM4, however, increased from 8.01 ± 0.56 of Mxyl to 12.5 ± 0.32 mg ml(-1), suggesting a decrease in the affinity as well as specific enzyme activity. The Mxyl as well as MxylM4 liberated chromophores and lignin-derived compounds from kraft pulp, indicating their applicability in pulp bleaching.

  15. 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.

  16. Enhanced Thermostability of Lipoxygenase from Anabaena sp. PCC 7120 by Site-Directed Mutagenesis Based on Computer-Aided Rational Design.

    Science.gov (United States)

    Diao, Hanwen; Zhang, Chong; Wang, Shuicheng; Lu, Fengxia; Lu, Zhaoxin

    2016-04-01

    Lipoxygenase from Anabaena sp. PCC 7120 (Ana-LOX) was thermally unstable. So, improving the thermostability of the enzyme was quite essential. The target site of Ana-LOX selected for site-directed mutagenesis was based on computer-aided rational design. The thermostability and specific activity of Ana-LOX were improved with replacing valine with alanine at the target site 421 and the site 40. Compared to the wild-type enzyme which has a half-life (T 1/2) of inactivation of 3.8 min at 50 °C, the T 1/2 of mutant enzymes with V421A and V40A substitution increased to 4.4 and 7.0 min, respectively. The double mutant V421A/V40A showed a synergistic effect with a T 1/2 value of 8.3 min, resulting in a 1.18-fold improvement compared to the original Ana-LOX. V421A, V40A, and V421A/V40A also obtained 4.83, 41.58, and 80.07 % increase in specific activity, respectively. This study provides useful theoretical reference for enzyme molecular modification and computer-aided rational design.

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

    Science.gov (United States)

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

    2015-11-01

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

  18. An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

    Institute of Scientific and Technical Information of China (English)

    Bao-zhong ZHANG; Xin ZHANG; Xiao-ping AN; Duo-liang RAN; Yu-sen ZHOU; Jun LU; Yi-gang TONG

    2009-01-01

    Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonuclease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5'-phosphorylated, circularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.

  19. Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase.

    Science.gov (United States)

    Kuno, T; Shuntoh, H; Sakaue, M; Saijoh, K; Takeda, T; Fukuda, K; Tanaka, C

    1988-06-30

    The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E. coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites. The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit. When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites. Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.

  20. Site-directed mutagenesis of amino acid residues of D1 protein interacting with phosphatidylglycerol affects the function of plastoquinone QB in photosystem II.

    Science.gov (United States)

    Endo, Kaichiro; Mizusawa, Naoki; Shen, Jian-Ren; Yamada, Masato; Tomo, Tatsuya; Komatsu, Hirohisa; Kobayashi, Masami; Kobayashi, Koichi; Wada, Hajime

    2015-12-01

    Recent X-ray crystallographic analysis of photosystem (PS) II at 1.9-Å resolution identified 20 lipid molecules in the complex, five of which are phosphatidylglycerol (PG). In this study, we mutagenized amino acid residues S232 and N234 of D1, which interact with two of the PG molecules (PG664 and PG694), by site-directed mutagenesis in Synechocystis sp. PCC 6803 to investigate the role of the interaction in PSII. The serine and asparagine residues at positions 232 and 234 from the N-terminus were mutagenized to alanine and aspartic acid, respectively, and a mutant carrying both amino acid substitutions was also produced. Although the obtained mutants, S232A, N234D, and S232AN234D, exhibited normal growth, they showed decreased photosynthetic activities and slower electron transport from QA to QB than the control strain. Thermoluminescence analysis suggested that this slower electron transfer in the mutants was caused by more negative redox potential of QB, but not in those of QA and S2. In addition, the levels of extrinsic proteins, PsbV and PsbU, were decreased in PSII monomer purified from the S232AN234D mutant, while that of Psb28 was increased. In the S232AN234D mutant, the content of PG in PSII was slightly decreased, whereas that of monogalactosyldiacylglycerol was increased compared with the control strain. These results suggest that the interactions of S232 and N234 with PG664 and PG694 are important to maintain the function of QB and to stabilize the binding of extrinsic proteins to PSII.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. The importance of Asn47 for structure and reactivity of azurin from Alcaligenes denitrificans as studied by site-directed mutagenesis and spectroscopy.

    Science.gov (United States)

    Hoitink, C W; Canters, G W

    1992-07-15

    To study the importance of a rigid copper site for the structure and function of azurin, a mutant with a reduced number of internal hydrogen bonds around the copper has been prepared and characterized. To this purpose, the previously cloned azu gene from Alcaligenes denitrificans (Hoitink, C. W. G., Woudt, L. P., Turenhout, J. C. M., Van de Kamp, M., and Canters, G. W. (1990) Gene (Amst.) 90, 15-20) was expressed in Escherichia coli and an isolation and purification procedure for the azurin was developed. The azurin obtained after heterologous expression in E. coli appears spectroscopically indistinguishable from azurin derived from A. denitrificans. The hydrogen bonding network around the copper site was altered by replacing Asn47 by a leucine by means of site-directed mutagenesis. Asn47 is a conserved residue in all blue copper proteins of which the primary structure has been reported. Characterization of the mutant protein with UV-visible, electron spin resonance, and NMR spectroscopy, and comparison with the wild type azurin revealed that the structure of the copper site as well as the overall structure of the protein have been largely retained. The redox activity as measured by the electron self-exchange rate appears not to have changed either. However, the mutant differs from the wild type azurin with respect to stability and midpoint potential. Midpoint potentials of mutant and wild type azurin amount to 396 and 286 mV, respectively. The difference is due to sizable entropic and enthalpic contributions which to a large extent cancel. Possible explanations for the outcome of these experiments are discussed.

  3. Site-directed mutagenesis reveals new and essential elements for iron-coordination of the sulfur oxygenasereductase from the acidothermophilic Acidianus teng-chongensis

    Institute of Scientific and Technical Information of China (English)

    CHEN ZhiWei; JIANG ChengYing; LIU ShuangJiang

    2009-01-01

    Previous study on refolding of sulfur oxygenase reductase (SOR) inclusion bodies from recombinant Escherichia coli showed that iron was critical to the activity of the SOR from Acidianus ambivalens. In this study, enzymatic assays showed that 2,2'-Dipyridyl, Tiron and 8-hydroxyquinoline, which are spe-cific for chelating ferrous or ferric ions, strongly inhibited the activity of SOR from A. tengchongensis, suggesting that iron atom is essential for SOR activity. Alignment of several functionally identified SORs and SOR-like sequences from genome database revealed a conserved, putative iron binding motif, H86-X3-H90-Xn-E114-Xn-E129 (numbering according to the Acidianus tengchongensis SOR sequence). Three mutants of SOR were generated by site-directed mutagenesis of H86, H90 and E129 into phenyla-lanine or alanine residue in this study. Circular dichroism spectrum determination indicated that there was no change of the secondary structures of mutant SORs, H86F, H90F and E129A, but all mutants were completely inactive. Through determination of iron contents we found that SOR mutants of H86F, H90F and E129A completely or partially lost iron, while mutants of C31S, C101S, and C104S (generated in a pre-vious study) did not. This result indicated that H86, H90 and E129 but not C31, C101, and C104 were involved in binding to iron atom. Based on this and previous studies, it is proposed that the conserved motifs, C31-Xn-C101-X2-C104 and H86-X3-H90-X23-E114-X14-(E/D)129, are respectively for sulfur and molecular oxygen binding and activation. These two conserved motifs are essential elements for the SOR activity.

  4. 计算机模建和点突变分析抗人CD40单克隆抗体5C11识别的抗原表位%Antigenic epitopes of anti-CD40 monoclonal antibody (5C11) analyzed by computer modeling and site-directed mutagenesis

    Institute of Scientific and Technical Information of China (English)

    张婷; 张学光; 瞿秋霞; 章良

    2011-01-01

    Objective; To primarily identify the antigenic epitopes of agonist type anti-CD40 monoclonal antibody, 5C11, which was constructed in our previous research, by means of computer modeling and site-directed mutation experiments. Methods: The structures of antigen and antibody were modeled by Insight II software and the immune complex was constructed. The antigenic epitope of 5C11 antibody was calculated and speculated. The full length of wide type human CD40 (wtCD40) gene and two site-directed mutant CD40 gene (70muCD40 and 114muCD40) were amplified by RT-PCR; pIRES2-EGFP/wtCD40, pIRES2-EGFP/70muCD40 and pIRES2-EGFP/114muCD40 recombinant vectors were constructed. These vectors were transfected into HEK293 cells by Lipofect method, and HEK293 cells stably transfected with pIRES2-EGFP/wtCD40, pIRES2-EGFP/70muCD40 and pIRES2-EGFP/l 14muCD40 vectors (named HEK293/ wtCD40, HEK293/70muCD40 and HEK293/114muCD40 cells, respectively) were screened. The binding abilities of HEK293/wtCD40, HEK293/70muCD40 and HEK293/114muCD40 cells with 5C11 were examined by flow cytometry and Western blotting analysis. Results: The recombinant eukaryotic expression vectors pIRES2-EGFP/wtCD40, pIRES2-EGFP/70muCD40 and pIRES2-EGFP/l 14muCD40 were successfully constructed and the corresponding stably transfected HEK293 cells were obtained. The binding ability between 5C11 antibody and HEK293/70muCD40 and HEK293/ 114muCD40 cells were lower than that with HEK293/wtCD40 cells. Western blotting results showed that 5C11 antibody only recognized HEK293/wtCD40 cells but not HEK293/70muCD40 and HEK293/114muCD40 cells. Conclusion; The 70' threonine and 114 glutamic acids in human CD40 amino acid sequence are the antigenic epitopes of 5C11 monoclonal antibody, which has potential clinical significance for humanized CD40 antibody research.%目的:通过计算机模拟与点突变实验初步探讨本课题组前期研制的抗人CD40激发型单克隆抗体5C11识别的抗原表位.方法:利用InsightⅡ

  5. 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

  6. Role of invariant tyrosines in a crustacean mu-class glutathione S-transferase from shrimp Litopenaeus vannamei: site-directed mutagenesis of Y7 and Y116.

    Science.gov (United States)

    Contreras-Vergara, Carmen A; Valenzuela-Soto, Elisa M; Arvizu-Flores, Aldo A; Sotelo-Mundo, Rogerio R; Yepiz-Plascencia, Gloria

    2008-06-01

    Y6 and Y115 are key amino acids involved in enzyme-substrate interactions in mu-class glutathione S-transferase (GST). They provide electrophilic assistance and stabilize substrates through their hydroxyl groups. Two site-directed mutants (Y7F and Y116F) and the wild-type shrimp GSTs were expressed in Escherichia coli, and the steady-state kinetic parameters were determined using CDNB as the second substrate. The mutants were modeled based on a crystal structure of a mu-class GST to obtain further insights about the changes at the active site. The Y116F mutant had an increase in kcat contrary to Y7F compared to the wild type. Molecular modeling showed that the shrimp GST has a H108 residue that may contribute to compensate and lead to a less deleterious change when conserved tyrosine residues are mutated. This work indicates that shrimp GST is a useful model to understand the catalysis mechanisms in this critical enzyme.

  7. Use of site-directed mutagenesis of allele-specific PCR primers to identify the GSTM1 A, GSTM1 B, GSTM1 A,B and GSTM1 null polymorphisms at the glutathione S-transferase, GSTM1 locus.

    OpenAIRE

    Fryer, A A; Zhao, L.; Alldersea, J; Pearson, W R; Strange, R C

    1993-01-01

    We describe the identification of the GSTM1 null, GSTM1 A, GSTM1 B and GSTM1 A,B polymorphisms at the glutathione S-transferase GSTM1 locus using a single-step PCR method. Target DNA was amplified using primers to intron 6 and exon 7 with site-directed mutagenesis being used to introduce a restriction site in DNA amplified from GSTM1 *A, thereby allowing differentiation of this allele and GSTM1 *B. The accuracy of this approach in identifying the GSTM1 A, GSTM1 B, GSTM1 A,B and GSTM1 null pol...

  8. Site-directed mutagenesis of the Klebsiella pneumoniae nifL and nifH promoters and in vivo analysis of promoter activity.

    OpenAIRE

    Buck, M; Khan, H.; Dixon, R

    1985-01-01

    The role of conserved nucleotides in nitrogen-fixation promoter function has been examined using both oligonucleotide and chemical mutagenesis to introduce base changes in the Klebsiella pneumoniae nifL and nifH promoters. Among ten mutations analysed, including six spontaneous mutations, base changes at -12, -13, -14, and -26, located in previously identified conserved sequences, perturbed the activity of the promoters, demonstrating that these sequences are required for transcription. Not a...

  9. A cell-based computational modeling approach for developing site-directed molecular probes.

    Directory of Open Access Journals (Sweden)

    Jing-Yu Yu

    Full Text Available Modeling the local absorption and retention patterns of membrane-permeant small molecules in a cellular context could facilitate development of site-directed chemical agents for bioimaging or therapeutic applications. Here, we present an integrative approach to this problem, combining in silico computational models, in vitro cell based assays and in vivo biodistribution studies. To target small molecule probes to the epithelial cells of the upper airways, a multiscale computational model of the lung was first used as a screening tool, in silico. Following virtual screening, cell monolayers differentiated on microfabricated pore arrays and multilayer cultures of primary human bronchial epithelial cells differentiated in an air-liquid interface were used to test the local absorption and intracellular retention patterns of selected probes, in vitro. Lastly, experiments involving visualization of bioimaging probe distribution in the lungs after local and systemic administration were used to test the relevance of computational models and cell-based assays, in vivo. The results of in vivo experiments were consistent with the results of in silico simulations, indicating that mitochondrial accumulation of membrane permeant, hydrophilic cations can be used to maximize local exposure and retention, specifically in the upper airways after intratracheal administration.

  10. Mapping the UDP-N-acetylglucosamine regulatory site of human glucosamine-6P synthase by saturation-transfer difference NMR and site-directed mutagenesis.

    Science.gov (United States)

    Assrir, Nadine; Richez, Celine; Durand, Philippe; Guittet, Eric; Badet, Bernard; Lescop, Ewen; Badet-Denisot, Marie-Ange

    2014-02-01

    The enzyme glucosamine-6P Synthase (Gfat, L-glutamine:D-fructose-6P amidotransferase) is involved in the hexosamine biosynthetic pathway and catalyzes the formation of glucosamine-6P from the substrates d-fructose-6-phosphate and l-glutamine. In eukaryotic cells, Gfat is inhibited by UDPGlcNAc, the end product of the biochemical pathway. In this work we present the dissection of the binding and inhibition properties of this feedback inhibitor and of its fragments by a combination of STD-NMR experiments and inhibition measurements on the wild type human enzyme (hGfat) as well as on site-directed mutants. We demonstrate that the UDPGlcNAc binding site is located in the isomerase domain of hGfat. Two amino acid residues (G445 and G461) located at the bottom of the binding site are identified to play a key role in the specificity of UDPGlcNAc inhibition of hGfat activity vs its bacterial Escherichia coli counterpart. We also show that UDPGlcNAc subcomponents have distinct features: the nucleotidic moiety is entirely responsible for binding whereas the N-acetyl group is mandatory for inhibition but not for binding, and the sugar moiety acts as a linker between the nucleotidic and N-acetyl groups. Combining these structural recognition determinants therefore appears as a promising strategy to selectively inhibit hGfat, which may for example help reduce complications in diabetes.

  11. Analysis of the nucleus-encoded and chloroplast-targeted rieske protein by classic and site-directed mutagenesis of Chlamydomonas.

    Science.gov (United States)

    de Vitry, C; Finazzi, G; Baymann, F; Kallas, T

    1999-10-01

    Three mutants of the alga Chlamydomonas reinhardtii affected in the nuclear PETC gene encoding the Rieske iron-sulfur protein 2Fe-2S subunit of the chloroplast cytochrome b(6)f complex have been characterized. One has a stable deletion that eliminates the protein; two others carry substitutions Y87D and W163R that result in low accumulation of the protein. Attenuated expression of the stromal protease ClpP increases accumulation and assembly into b(6)f complexes of the Y87D and W163R mutant Rieske proteins in quantities sufficient for analysis. Electron-transfer kinetics of these complexes were 10- to 20-fold slower than those for the wild type. The deletion mutant was used as a recipient for site-directed mutant petC alleles. Six glycine residues were replaced by alanine residues (6G6A) in the flexible hinge that is critical for domain movement; substitutions were created near the 2Fe-2S cluster (S128 and W163); and seven C-terminal residues were deleted (G171och). Although the 6G6A and G171och mutations affect highly conserved segments in the chloroplast Rieske protein, photosynthesis in the mutants was similar to that of the wild type. These results establish the basis for mutational analysis of the nuclear-encoded and chloroplast-targeted Rieske protein of photosynthesis.

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

    Science.gov (United States)

    Carroll, Thomas M.; Setlow, Peter

    2005-01-01

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

  13. 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)

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Analysis of an invariant cofactor-protein interaction in thiamin diphosphate-dependent enzymes by site-directed mutagenesis. Glutamic acid 418 in transketolase is essential for catalysis.

    Science.gov (United States)

    Wikner, C; Meshalkina, L; Nilsson, U; Nikkola, M; Lindqvist, Y; Sundström, M; Schneider, G

    1994-12-23

    A homologous expression system and a purification protocol for pure, highly active recombinant yeast transketolase have been developed. The invariant transketolase residue Glu418, which forms a hydrogen bond to the N-1' nitrogen atom of the pyrimidine ring of the cofactor thiamin diphosphate has been replaced by glutamine and alanine. Crystallographic analyses of the mutants show that these amino acid substitutions do not induce structural changes beyond the site of mutation. In both cases, the cofactor binds in a manner identical to the wild-type enzyme. Significant differences in the CD spectra of the mutant transketolases compared with the spectrum of wild-type enzyme indicate differences in the electron distribution of the aminopyrimidine ring of the cofactor. The E418Q mutant shows 2% and the E418A mutant shows about 0.1% of the catalytic activity of wild-type enzyme. The affinities of the mutant enzymes for thiamin diphosphate are comparable with wild-type transketolase. The hydrogen bond between the coenzyme and the side chain of Glu418 is thus not required for coenzyme binding but essential for catalytic activity. The results demonstrate the functional importance of this interaction and support the molecular model for cofactor deprotonation, the first step in enzymatic thiamin catalysis.

  16. Site-directed Mutagenesis Shows the Significance of Interactions with Phospholipids and the G-protein OsYchF1 for the Physiological Functions of the Rice GTPase-activating Protein 1 (OsGAP1).

    Science.gov (United States)

    Yung, Yuk-Lin; Cheung, Ming-Yan; Miao, Rui; Fong, Yu-Hang; Li, Kwan-Pok; Yu, Mei-Hui; Chye, Mee-Len; Wong, Kam-Bo; Lam, Hon-Ming

    2015-09-25

    The C2 domain is one of the most diverse phospholipid-binding domains mediating cellular signaling. One group of C2-domain proteins are plant-specific and are characterized by their small sizes and simple structures. We have previously reported that a member of this group, OsGAP1, is able to alleviate salt stress and stimulate defense responses, and bind to both phospholipids and an unconventional G-protein, OsYchF1. Here we solved the crystal structure of OsGAP1 to a resolution of 1.63 Å. Using site-directed mutagenesis, we successfully differentiated between the clusters of surface residues that are required for binding to phospholipids versus OsYchF1, which, in turn, is critical for its role in stimulating defense responses. On the other hand, the ability to alleviate salt stress by OsGAP1 is dependent only on its ability to bind OsYchF1 and is independent of its phospholipid-binding activity.

  17. 利用定点突变法研究精氨酸脱亚胺酶活性的影响机制%Mechanism of arginine deiminase activity by site-directed mutagenesis

    Institute of Scientific and Technical Information of China (English)

    李利锋; 倪晔; 孙志浩

    2012-01-01

    精氨酸脱亚胺酶(ADI)是一种针对精氨酸缺陷型癌症(如:肝癌、黑素瘤)的新药,目前处于临床三期试验.文中通过定点突变技术分析了精氨酸脱亚胺酶的特定氨基酸位点对酶活力的影响机制.针对已报道的关键氨基酸残基A128、H404、I410,采用QuikChange法进行定点突变,获得ADI突变株M1 (A128T)、M2 (H404R)、M3 (I410L)和M4 (A128T/H404R).将突变株在大肠杆菌BL21 (DE3)中进行重组表达,并对纯化获得的突变蛋白进行酶学性质研究.结果表明,突变位点A128T和H404R对ADI最适pH的提高,生理中性(pH 7.4)条件下的酶活力和稳定性的提高,以及Km值的降低均具有显著的作用.研究结果为阐明ADI的酶活力影响机制和蛋白质的理性改造提供了一定的依据.%Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants Ml (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced Km value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-11-01

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

  19. Improvement of Thermostability of Penicillium expansum Lipase by Site-directed Mutagenesis%定点突变对扩展青霉脂肪酶热稳定性的改善

    Institute of Scientific and Technical Information of China (English)

    蔡少丽; 邹有土; 黄建忠; 林琳

    2013-01-01

    利用重叠延伸PCR方法对扩展青霉脂肪酶基因进行体外定点突变,构建了K115R与随机突变体ep8双突变的重组质粒pAO815-ep8-K115R.将该质粒电转化到毕赤酵母GS115中进行异源表达,获得双突变体脂肪酶PEL-ep8-K115R-GS.与野生型脂肪酶PEL-GS和随机突变体脂肪酶PEL-ep8-GS进行比较,发现双突变体脂肪酶PEL-ep8-K115R-GS在40℃温浴30 min后残余酶活为89%,比野生型脂肪酶PEL-GS和随机突变体脂肪酶PEL-ep8-GS分别提高54%和27%;双突变体脂肪酶PEL-ep8-K115R-GS的Tm值为42.2℃,比野生型脂肪酶PEL-GS提高3.5℃,比随机突变脂肪酶PEL-ep8-GS 提高2.0℃.研究结果表明对扩展青霉脂肪酶的分子改造能提高其热稳定性.%In order to improve the thermostability of Penicillium expansum lipase (PEL), the lipase gene was mutated by site-directed mutagenesis. A recombinant plasmid pAO815-ep8-K115R which contains double mutant genes was constructed by overlap extension PCR using the cDNA of a random-mutant lipase ep8 (a single site mutant) as the template and two special primers were used to generate another mutation site K115R. The recombinant vector was transformed into Pichia pastoris GS115 by electroporation and the recombinant mutant GS-pAO815-ep8-K115R can secret the double-mutant lipase PEL-ep8-K115R-GS into the medium when it was induced by methanol. Thermostability analysis revealed that the residual activity of the double-mutant lipase PEL-ep8-K115R-GS after incubated at 40 ℃ for 30 min was 54% and 27% higher than that of the wild type lipase PEL-GS and the random-mutant lipase PEL-ep8-GS respectively. Tm of the double-mutant lipase PEL-ep8-K115R-GS was 42.2 ℃, 3.5 ℃ higher than that of the wild type lipase PEL-GS, and 2.0 ℃ higher than that of the random-mutant lipase PEL-ep8-GS.Fig5,Ref 20

  20. Site-directed spectroscopic probes of actomyosin structural dynamics.

    Science.gov (United States)

    Thomas, David D; Kast, David; Korman, Vicci L

    2009-01-01

    Spectroscopy of myosin and actin has entered a golden age. High-resolution crystal structures of isolated actin and myosin have been used to construct detailed models for the dynamic actomyosin interactions that move muscle. Improved protein mutagenesis and expression technologies have facilitated site-directed labeling with fluorescent and spin probes. Spectroscopic instrumentation has achieved impressive advances in sensitivity and resolution. Here we highlight the contributions of site-directed spectroscopic probes to understanding the structural dynamics of myosin II and its actin complexes in solution and muscle fibers. We emphasize studies that probe directly the movements of structural elements within the myosin catalytic and light-chain domains, and changes in the dynamics of both actin and myosin due to their alternating strong and weak interactions in the ATPase cycle. A moving picture emerges in which single biochemical states produce multiple structural states, and transitions between states of order and dynamic disorder power the actomyosin engine.

  1. Site-directed mutagenesis of an HLA-A3 gene identifies amino acid 152 as crucial for major-histocompatibility-complex-restricted and alloreactive cytotoxic-T-lymphocyte recognition.

    OpenAIRE

    Cowan, E P; Jelachich, M L; Coligan, J E; Biddison, W E

    1987-01-01

    Major histocompatibility complex-restricted and alloreactive cytotoxic T lymphocytes (CTL) can discriminate between the HLA-A3.1 and HLA-A3.2 antigens. HLA-A3.1 and the rare variant HLA-A3.2 have been shown to differ by two amino acids in the alpha 2 domain at positions 152 (A3.1, glutamic acid; A3.2, valine) and 156 (A3.1, leucine; A3.2, glutamine). To determine the structural basis for the ability of CTL to differentiate A3.1 from A3.2, two site-directed mutants of the HLA-A3.2 gene were pr...

  2. TALEN-mediated somatic mutagenesis in murine models of cancer.

    Science.gov (United States)

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

    2014-09-15

    Cancer genome sequencing has identified numerous somatic mutations whose biologic relevance is uncertain. In this study, we used genome-editing tools to create and analyze targeted somatic mutations in murine models of liver cancer. Transcription activator-like effector nucleases (TALEN) were designed against β-catenin (Ctnnb1) and adenomatous polyposis coli (Apc), two commonly mutated genes in hepatocellular carcinoma (HCC), to generate isogenic HCC cell lines. Both mutant cell lines exhibited evidence of Wnt pathway dysregulation. We asked whether these TALENs could create targeted somatic mutations after hydrodynamic transfection into mouse liver. TALENs targeting β-catenin promoted endogenous HCC carrying the intended gain-of-function mutations. However, TALENs targeting Apc were not as efficient in inducing in vivo homozygous loss-of-function mutations. We hypothesized that hepatocyte polyploidy might be protective against TALEN-induced loss of heterozygosity, and indeed Apc gene editing was less efficient in tetraploid than in diploid hepatocytes. To increase efficiency, we administered adenoviral Apc TALENs and found that we could achieve a higher mutagenesis rate in vivo. Our results demonstrate that genome-editing tools can enable the in vivo study of cancer genes and faithfully recapitulate the mosaic nature of mutagenesis in mouse cancer models. Cancer Res; 74(18); 5311-21. ©2014 AACR.

  3. Cloning of the genomes of human cytomegalovirus strains Toledo, TownevarRIT3, and Towne long as BACs and site-directed mutagenesis using a PCR-based technique.

    Science.gov (United States)

    Hahn, Gabriele; Rose, Dietlind; Wagner, Markus; Rhiel, Sylvia; McVoy, Michael A

    2003-03-01

    The 230-kb human cytomegalovirus genome is among the largest of the known viruses. Experiments to determine the genetic determinants of attenuation, pathogenesis, and tissue tropism are underway; however, a lack of complete sequence data for multiple strains and substantial problems with genetic instability during in vitro propagation create serious complications for such studies. For example, recent findings suggest that common laboratory strains Towne and AD169 passaged in cultured human fibroblasts are missing up to 15 kb of genetic information relative to clinical isolates. To establish standard, genetically stable genomes that can be sequenced, disseminated, and repeatedly reconstituted to produce virus stocks, we have undertaken to clone two variants of Towne, designated Towne(long) and Towne(short) (referred to as TownevarRIT3) (A., Proc. Natl. Acad. Sci. USA 98, 7829-7834), and the pathogenic strain Toledo into bacterial artificial chromosomes (BACs). We further demonstrate the ease with which mutagenesis can be achieved by deleting 13.5 kb from the Toledo genome using a PCR-based technique.

  4. 用DREAM技术纠正人工合成基因中的突变%Correction of a Mutation in a Synthetic Gene by DREAM Technique, a Site-directed Mutagenesis

    Institute of Scientific and Technical Information of China (English)

    朱晓峰; 陈锦辉; 张昕; 安小平; 周育森; 童贻刚

    2007-01-01

    目的:介绍一种简便、有效的定点突变技术.方法:根据突变位点附近的DNA序列推导出氨基酸序列,再以此氨基酸序列进行逆翻译,这样在不改变氨基酸序列的前提下可以得到数目巨大的隐性突变(silent mutations),这些突变中包含大量的限制性内切酶位点,选择合适的酶切位点,自酶切位点向两侧合成引物(其中仅一个引物含有突变),用这些引物扩增两侧DNA片段(其中仅一个片段含有突变),然后将这两个片段以相应的内切酶切割后融合即可完成定点突变.结果:用该方法成功地纠正了人工合成的可溶性组织因子基因中两个碱基的缺失,校正了阅读框架,获得了预期的目的基因.结论:该方法简便、有效,突变成功率高,适合于在一般分子生物学实验室使用;该方法能避免了因多轮PCR和合成长引物导致突变的可能性,可作为一种定点突变的替代方法.这种改进的PCR定点诱变技术我们称之为"设计限制酶辅助突变"(Designed Restriction Enzyme Assisted Mutagenesis,DREAM).

  5. Contributions of tryptophan 24 and glutamate 30 to binding long-lived water molecules in the ternary complex of human dihydrofolate reductase with methotrexate and NADPH studied by site-directed mutagenesis and nuclear magnetic resonance spectroscopy.

    Science.gov (United States)

    Meiering, E M; Li, H; Delcamp, T J; Freisheim, J H; Wagner, G

    1995-03-24

    Previous NMR studies on the ternary complex of human dihydrofolate reductase (hDHFR) with methotrexate (MTX) and NADPH detected six long-lived bound water molecules. Two of the water molecules, WatA and WatB, stabilize the structure of the protein while the other four, WatC, WatD, WatE and WatF, are involved in substrate binding and specificity. WatE may also act as a proton shuttle during catalysis. Here, the contributions of individual residues to the binding of these water molecules are investigated by performing NMR experiments on ternary complexes of mutant enzymes, W24F, E30A and E30Q. W24 and E30 are conserved residues that form hydrogen bonds with WatE in crystal structures of DHFR. Nuclear Overhauser effects (NOEs) are detected between WatE and the protein in all the mutant complexes, hence WatE still has a long lifetime bound to the complex when one of its hydrogen-bonding partners is deleted or altered by mutagenesis. The NOEs for WatE are much weaker, however, in the mutants than in wild-type. The NOEs for the other water molecules in and near the active site, WatA, WatC, WatD and WatF, also tend to be weaker in the mutant complexes. Little or no change is apparent in the NOEs for WatB, which is located outside the active site, farthest from the mutated residues. The decreased NOE intensities for the bound water molecules could be caused by changes in the positions and/or lifetimes of the water molecules. Chemical shift and NOE data indicate that the mutants have structures very similar to that of wild-type hDHFR, with possible conformational changes occurring only near the mutated residues. Based on the lack of structural change in the protein and evidence for increased structural fluctuations in the active sites of the mutant enzymes, it is likely that the NOE changes are caused, at least in part, by decreases in the lifetimes of the bound water molecules.

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

    Science.gov (United States)

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

    2015-01-01

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

  7. Stationary-State Mutagenesis in Escherichia coli: a Model

    Indian Academy of Sciences (India)

    S. K. Mahajan; A. V. S. S. Narayana Rao; S. K. Bhattacharjee

    2000-04-01

    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 mechanism that generates a transient state of hypermutagenesis, operating on a large number of sites spread over the entire genome, at least in a proportion of the resting cells. Most of the studies that clarified this position were on the reversion of a frameshift mutation present in a lacI -- lacZ fusion in E. coli strain FC40. Several groups have extensively examined both the sequence changes associated with these reversions and the underlying genetic requirements. On the basis of our studies on the genomic sequence analysis, we recently proposed a model to explain the specific changes associated with the reversion hotspots. Here we propose a more detailed version of this model that also takes into account the observed genetic requirements of stationary-state mutagenesis. Briefly, G:T/U mismatches produced at methylatable cytosines are preferentially repaired in nondividing cells by the very short patch mismatch repair (VSPMR) mechanism which is itself mutagenic and can produce mutations in very short stretches located in the immediate vicinity of these cytosine methylation sites. This mechanism requires a homologous or homeologous strand invasion step and an error-prone DNA synthesis step and is dependent on RecA, RecBCD and a DNA polymerase. The process is initiated near sequences recognized by Dcm and Vsr enzymes and further stimulated if these sequences are a part of CHI or CHI-like sequences, but a double-strand-break-dependent recombination mediated by the RecBCD pathways proposed by others seems to be nonessential. The strand transfer step is proposed to depend on RecA, RuvA, RuvB and RuvC and is opposed by RecG and MutS. The model also gives

  8. Understanding microscopic binding of human microsomal prostaglandin E synthase-1 (mPGES-1) trimer with substrate PGH2 and cofactor GSH: insights from computational alanine scanning and site-directed mutagenesis.

    Science.gov (United States)

    Hamza, Adel; Tong, Min; AbdulHameed, Mohamed Diwan M; Liu, Junjun; Goren, Alan C; Tai, Hsin-Hsiung; Zhan, Chang-Guo

    2010-04-29

    Microsomal prostaglandin E synthase-1 (mPGES-1) is an essential enzyme involved in a variety of diseases and is the most promising target for the design of next-generation anti-inflammatory drugs. In order to establish a solid structural base, we recently developed a model of mPGES-1 trimer structure by using available crystal structures of both microsomal glutathione transferase-1 (MGST1) and ba3-cytochrome c oxidase as templates. The mPGES-1 trimer model has been used in the present study to examine the detailed binding of mPGES-1 trimer with substrate PGH(2) and cofactor GSH. Results obtained from the computational alanine scanning reveal the contribution of each residue at the protein-ligand interaction interface to the binding affinity, and the computational predictions are supported by the data obtained from the corresponding wet experimental tests. We have also compared our mPGES-1 trimer model with other available 3D models, including an alternative homology model and a low-resolution crystal structure, and found that our mPGES-1 trimer model based on the crystal structures of both MGST1 and ba3-cytochrome c oxidase is more reasonable than the other homology model of mPGES-1 trimer constructed by simply using a low-resolution crystal structure of MGST1 trimer alone as a template. The available low-resolution crystal structure of mPGES-1 trimer represents a closed conformation of the enzyme and thus is not suitable for studying mPGES-1 binding with ligands. Our mPGES-1 trimer model represents a reasonable open conformation of the enzyme and is therefore promising for studying mPGES-1 binding with ligands in future structure-based drug design targeting mPGES-1.

  9. 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.

  10. 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.

  11. CRISPR/Cas9技术在HepG2基因组中进行定点基因突变的探索%Exploration of site-directed mutagenesis mediated by CRISPR/Cas9 system in HepG2

    Institute of Scientific and Technical Information of China (English)

    肖利佳; 龙寿斌; 罗历; 沈化清; 郝建华

    2015-01-01

    Objective To explore whether CRISPR/Cas9 system could be effective in hepatocarcinoma cell line HepG2. Methods gRNA plasmids targeting nuclear receptor LRH-1 and ERRαwere designed and con-structed. After verification by sequencing, gRNA-LRH-1/gRNA-ERRα and hCas9 plasmids were transfected in HepG2 cells respectively. Then mutation sites were amplified by PCR and mutation rates were evaluated by SUR-VEYOR assay. Results gRNA-LRH-1 and gRNA-ERRαrecombination plasmids targeting nuclear receptor LRH-1 and ERRαwere successfully established. After transfecting HepG2 with gRNA-LRH-1/gRNA-ERRαand hCas9, the mutation sites were amplified by PCR and subjected to SURVEYOR assay. The electrophoresis results showed that two additional bands with expected size were found in cells transfected with gRNA-LRH-1/gRNA-ERRαand hCAS9. Conclusion The site-directed mutagenesis of LRH-1 and ERRαgene locus were successfully created by genome ed-iting mediated by CRISPR/Cas9 in HepG2, which provides the basis for the generation of HepG2-LRH-1/ERRαknockout cell model.%目的:探索利用CRISPR/Cas9技术在细胞株HepG2基因组中进行定点突变。方法设计并构建靶向核受体LRH-1和ERRα基因组序列的gRNA质粒。将构建好的gRNA质粒和hCas9质粒转染HepG2细胞后,PCR扩增HepG2基因组中LRH-1和ERRα基因的突变位点,并用SURVEYOR法检测突变情况。结果靶向核受体LRH-1和ERRα基因组序列的gRNA质粒构建成功。HepG2细胞经gRNA-LRH-1/gRNA-ERRα和hCas9转染后,针对LRH-1和ERRα的突变位点基因组序列的PCR产物经SURVEYOR法检测结果显示出现两条与预计大小相符的电泳条带。结论在HepG2细胞中成功利用CRISPR/Cas9技术介导的基因组编辑对LRH-1和ERRα特定基因组序列产生突变,为构建其细胞株敲除模型奠定了基础。

  12. Construction of plasmid containing IVS-2-654 (C>T) single mutation of β-thalassemia gene by site-directed mutagenesis%定点诱变法构建β地中海贫血IVS-2-654(C>T)突变基因质粒

    Institute of Scientific and Technical Information of China (English)

    余玲玲; 田可港; 冯晶晶; 王慧燕; 郑晓群

    2012-01-01

    Objective To construct a plasmid containing IVS-2-654 (OT) single site mutation of p-thalassemia gene. Methods The plasmid DNA containing wild-type β-globin gene was used as PCR template. Plasmid containing IVS-2-654 (OT) mutation gene of β-globin was constructed by TA clone technology after site-directed mutagenesis of overlap extension PCR (OE-PCR). Results Direct DNA sequencing showed that the recombi-nant plasmid contained IVS-2-654 (OT) mutation gene of β-globin, which mutated from C to T at IVS-2-654 bp and the rest was completely identical with wide type. Conclusions The plasmid containing IVS-2-654 (C> T) mutation gene was successfully constructed, which laied the foundation for further screening studies to detect gene mutation of (Hhalassemia and other genetic diagnosis for this disease. Site-directed mutagenesisis of OE-PCR technique is simple and economic and worth of being promoted widely.%目的 构建含β地中海贫血(简称β地贫)IVS-2-654 (C>T)突变基因的质粒.方法 以含β珠蛋白野生型基因的质粒DNA为模板,采用重叠延伸PCR (OE-PCR)定点诱变后行TA克隆的方法构建含IVS-2-654 (C>T)突变基因的质粒.结果 双向测序结果表明:重组质粒的确含β地贫IVS-2-654 (C>T)突变基因,β珠蛋白IVS-2-654处的碱基已由C突变成T,其余序列与野生型完全相同,成功实现了定点诱变.结论 成功地构建了含β地贫IVS-2-654 (C>T)突变基因的质粒,进一步为该病基因诊断与筛查技术的研究奠定了实验基础;OE-PCR定点诱变法简便、经济,值得推广应用.

  13. 定向诱变方法研究结核分支杆菌KatG基因突变与异烟肼耐药机制的关系%The effect of point mutation of KatG gene on the catalase activity associated with INH resistance by site-directed mutagenesis

    Institute of Scientific and Technical Information of China (English)

    张文宏; 陈澍; 季朝能; 庞茂银; 邵凌云; 华正豪; 翁心华

    2001-01-01

    目的探讨结核分支杆菌KatG基因的常见点突变是否会导致与产生异烟肼耐药性相关的过氧化氢酶活性降低.方法用定向诱变方法产生耐异烟肼的结核分支杆菌中最常见的KatG基因315突变体,造成该位点从丝氨酸(AGC)到苏氨酸(ACC)的突变.随后构建含KatG基因S315T突变体的质粒,转化进入大肠杆菌并实现高表达,对表达的蛋白进行过氧化氢酶活性的测定.结果通过定向诱变方法成功获得KatG基因S315T突变体,并通过pET24b质粒转入大肠杆菌,Ka tG基因S315T突变体蛋白在大肠杆菌中得到高表达.对KatG S315T表达产物的过氧化氢酶活性进行检测,发现比野生株KatG基因表达产物酶活性下降了50%左右.结论KatG基因315位从丝氨酸(AGC)到苏氨酸(ACC)的突变造成过与异烟肼耐药产生直接相关的氧化氢酶活性的下降.%Objective To determine whether specific missense mutations in the M. Tuberculosis katG gene could result in a loss of enzymatic activity associated with resistance to isoniazid (INH).Methods The authors used site-directed mutagenesis to induce the commonest mutation S315T in the KatG gene of INH-resistant M. Tuberculosis. Furthermore, we constructed a plasmid containing katG with the S315T mutation, expressed the recombinant protein in a catalase-peroxidase-deficient strain of Escherichia coli, and assessed its enzymatic activity via detecting the release of O2 from whole-cell organisms.Results The mutated katG gene S315T was successfully obtained by site-directed mutagenesis and constructed in pET24b and transformed into E. Coli. The katG(S315T)protein was then expressed and the catalase activities of which were detected. It was found that the catalase activity of S315T katG mutant was reduced 50% compared with KatG (wt).Conclusions The results suggest that S315T leads to reducing the activity of catalase which is related with activating INH. The method to detect the gene function of Kat

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

    Science.gov (United States)

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

    2013-08-01

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

  15. Ligand binding modes from low resolution GPCR models and mutagenesis: chicken bitter taste receptor as a test-case.

    Science.gov (United States)

    Di Pizio, Antonella; Kruetzfeldt, Louisa-Marie; Cheled-Shoval, Shira; Meyerhof, Wolfgang; Behrens, Maik; Niv, Masha Y

    2017-08-15

    Bitter taste is one of the basic taste modalities, warning against consuming potential poisons. Bitter compounds activate members of the bitter taste receptor (Tas2r) subfamily of G protein-coupled receptors (GPCRs). The number of functional Tas2rs is species-dependent. Chickens represent an intriguing minimalistic model, because they detect the bitter taste of structurally different molecules with merely three bitter taste receptor subtypes. We investigated the binding modes of several known agonists of a representative chicken bitter taste receptor, ggTas2r1. Because of low sequence similarity between ggTas2r1 and crystallized GPCRs (~10% identity, ~30% similarity at most), the combination of computational approaches with site-directed mutagenesis was used to characterize the agonist-bound conformation of ggTas2r1 binding site between TMs 3, 5, 6 and 7. We found that the ligand interactions with N93 in TM3 and/or N247 in TM5, combined with hydrophobic contacts, are typically involved in agonist recognition. Next, the ggTas2r1 structural model was successfully used to identify three quinine analogues (epiquinidine, ethylhydrocupreine, quinidine) as new ggTas2r1 agonists. The integrated approach validated here may be applicable to additional cases where the sequence identity of the GPCR of interest and the existing experimental structures is low.

  16. 先天性QT间期延长综合征相关基因SCN5A定点突变及蛋白表达研究%Site-directed mutagenesis and protein expression of SCN5A gene associated with congenital Long QT syndrome

    Institute of Scientific and Technical Information of China (English)

    史瑞明; 强华; 张艳敏; 马爱群; 高洁

    2013-01-01

    目的 构建先天性QT间期延长综合征相关基因SCN5A-delQKP1507-1509突变型真核表达载体,并观察其在人胚肾293(HEK293)细胞中的表达.方法 采用一步法构建SCN5 A-delQKP1507-1509突变型真核表达载体PEGFP-delQKP-hH1,用脂质体转染法将野生型和突变型质粒分别转染HEK293细胞,激光共聚焦显微镜观察钠通道蛋白在HEK293细胞的表达与定位,Western blot检测其蛋白表达.结果 经电泳及DNA测序显示突变型1507-1509位点成功缺失9个碱基,野生型与突变型均在HEK293细胞膜上表达,且表达量差异无统计学意义(P>0.05).结论 成功构建钠通道基因SCN5 A-delQKP 1507-1509突变型真核表达载体,并在HEK293细胞中表达,为进一步研究其功能奠定基础.%Objective To construct the sodium channel gene SCN5A-delQKP1507-1509 mutation associated with congenital long QT syndrome, and its eukaryotic expression vector, and to examine the expression of mutation protein in human embryonic kidney (HEK) 293 cells. Methods Eukaryotic expression vector PEGFP-delQKP-hHl for SCN5A-delQKP1507-1509 mutation was constructed by rapid site-directed mutagenesis. HEK293 cells were transfected with the wild or mutant vector using lipofectamine, and then subjected to confocal microscopy. The transfected cells were immunostained to visualize intracellular expression of the mutant molecules. Results Direct sequence and electrophoresis analysis revealed 9 basic group absences at position 1507-1509. The delQKP1507-1509 mutation eukaryotic expression vector was expressed in HEK293 cells. Immunostaining of transfected cells showed the expression of both wild type and mutant molecules on the plasma membrane and there was no difference in the amount of protein, which suggested that the mutant delQKP1507-1509 did not impair normal protein expression in HEK293 cells. Conclusions Successful construction of mutant SCN5AdelQKP1507-1509 eukaryotic expression vector and expression of SCN5A

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

    Science.gov (United States)

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

    1992-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sandeep Krishna

    2007-03-01

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

  19. Genotoxin Induced Mutagenesis in the Model Plant Physcomitrella patens

    Directory of Open Access Journals (Sweden)

    Marcela Holá

    2013-01-01

    Full Text Available The moss Physcomitrella patens is unique for the high frequency of homologous recombination, haploid state, and filamentous growth during early stages of the vegetative growth, which makes it an excellent model plant to study DNA damage responses. We used single cell gel electrophoresis (comet assay to determine kinetics of response to Bleomycin induced DNA oxidative damage and single and double strand breaks in wild type and mutant lig4 Physcomitrella lines. Moreover, APT gene when inactivated by induced mutations was used as selectable marker to ascertain mutational background at nucleotide level by sequencing of the APT locus. We show that extensive repair of DSBs occurs also in the absence of the functional LIG4, whereas repair of SSBs is seriously compromised. From analysis of induced mutations we conclude that their accumulation rather than remaining lesions in DNA and blocking progression through cell cycle is incompatible with normal plant growth and development and leads to sensitive phenotype.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Alex S Nord

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

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

    Directory of Open Access Journals (Sweden)

    Hill Colin

    2010-12-01

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

  3. 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

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    Chen Tsung-Chieh

    2012-05-01

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

  6. 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

    2016-08-18

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

  7. Study on the site-directed mutagenesis,expression and purification of HPV 16E7 oncogenic sites%H PV16E7致瘤相关位点的定向突变及其表达纯化

    Institute of Scientific and Technical Information of China (English)

    彭小丽; 郭龙华; 吴文权; 刘梦琼

    2016-01-01

    Objective To explore the related sites of transformation in site‐directed mutagenesis of HPV16E7 gene and to study the expression and immunogenicity after the mutation of HPV 16E7 in prokaryotic cell .Methods Mutated sites were designed in the primer .Full length HPV16E7 gene were cloned by T carrier .Then the plasmid of pET‐28a‐HPV16E7 was subcloned (named pET‐28a‐HPVm16E7) ,and the bacteria of E .Coli BL21 were transformed and induced by isopropyl thiogalactoside (ITPG) to ex‐press HPVm16E7 protein .The protein was analyzed by Western Blot using monoclonal antibody of HPV 16E7 .Results DNA se‐quencing showed that pET‐28a‐HPVm16E7 was direct .The bacteria after transformation could express HPVm16E7 protein and re‐act with different kinds of HPV16E7 monoclonal antibodies .Conclusion Mutated sites designed in the primer can make the muta‐tion and transformation of HPV16E7 gene directionally .Expression of HPVm16E7 in prokaryotic cells would not influence the im‐munogenicity while increase the security of HPV16E7 vaccine .%目的:研究定点突变 HPV16E7基因中与转化有关的位点,原核细胞表达突变后的 HPV16E7的表达及其免疫原性。方法在引物内设计突变位点,T载体克隆全部与转化有关的全长 HPV16E7基因,然后亚克隆构建pET‐28a‐HPV16E7质粒(命名为pET‐28a‐HPVm16E7),转化 E .Coli BL21细菌,在异丙基硫代半乳糖苷(IPTG )诱导下表达 HPVm16E7蛋白,用HPV16E7单克隆抗体对其进行Western Blot分析。结果 DNA测序证明了构建的pET‐28a‐HPVm16E7是正确的,转化细菌后能够表达HPVm16E7蛋白,并能与不同的HPV16E7单抗抗体反应。结论在引物中设计突变位点能够定向突变 HPV16E7与转化有关的位点,原核表达的HPVm16E7不影响其免疫原性,增加了基于 HPV16E7的疫苗的安全性。

  8. Modeling nucleotide excision repair and its impact on UV-induced mutagenesis during SOS-response in bacterial cells.

    Science.gov (United States)

    Bugay, Aleksandr N; Krasavin, Evgeny A; Parkhomenko, Aleksandr Yu; Vasilyeva, Maria A

    2015-01-01

    A model of the UV-induced mutation process in Escherichia coli bacteria has been developed taking into account the whole sequence of molecular events starting from initial photo-damage and finishing with the fixation of point mutations. The wild-type phenotype bacterial cells are compared with UV-sensitive repair-deficient mutant cells. Attention is mainly paid to excision repair system functioning as regards induced mutagenesis.

  9. 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.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2014-05-01

    The ice binding motifs of insect antifreeze proteins (AFPs) mainly consist of repetitive TxT motifs aligned on a flat face of the protein. However, these motifs often contain non-threonines that disrupt the TxT pattern. We substituted two such disruptive amino acids located in the ice binding face of an AFP from Rhagium mordax with threonine. Furthermore, a mutant with an extra ice facing TxT motif was constructed. These mutants showed enhanced antifreeze activity compared to the wild type at low concentrations. However, extrapolating the data indicates that the wild type will become the most active at concentrations above 270 μmol.

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

    Science.gov (United States)

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

    2007-01-01

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

  13. E. coli F1-ATPase: site-directed mutagenesis of the beta-subunit.

    Science.gov (United States)

    Parsonage, D; Wilke-Mounts, S; Senior, A E

    1988-05-09

    Residues beta Glu-181 and beta Glu-192 of E. coli F1-ATPase (the DCCD-reactive residues) were mutated to Gln. Purified beta Gln-181 F1 showed 7-fold impairment of 'unisite' Pi formation from ATP and a large decrease in affinity for ATP. Thus the beta-181 carboxyl group in normal F1 significantly contributes to catalytic site properties. Also, positive catalytic site cooperativity was attenuated from 5 X 10(4)- to 548-fold in beta Gln-181 F1. In contrast, purified beta Gln-192 F1 showed only 6-fold reduction in 'multisite' ATPase activity. Residues beta Gly-149 and beta Gly-154 were mutated to Ile singly and in combination. These mutations, affecting residues which are strongly conserved in nucleotide-binding proteins, were chosen to hinder conformational motion in a putative 'flexible loop' in beta-subunit. Impairment of purified F1-ATPase ranged from 5 to 61%, with the double mutant F1 less impaired than either single mutant. F1 preparations containing beta Ile-154 showed 2-fold activation after release from membranes, suggesting association with F0 restrained turnover on F1 in these mutants.

  14. 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.

  15. Site-directed mutagenesis of aldehyde dehydrogenase-2 suggests three distinct pathways of nitroglycerin biotransformation.

    Science.gov (United States)

    Wenzl, M Verena; Beretta, Matteo; Griesberger, Martina; Russwurm, Michael; Koesling, Doris; Schmidt, Kurt; Mayer, Bernd; Gorren, Antonius C F

    2011-08-01

    To elucidate the mechanism underlying reduction of nitroglycerin (GTN) to nitric oxide (NO) by mitochondrial aldehyde dehydrogenase (ALDH2), we generated mutants of the enzyme lacking the cysteines adjacent to reactive Cys302 (C301S and C303S), the glutamate that participates as a general base in aldehyde oxidation (E268Q) or combinations of these residues. The mutants were characterized regarding acetaldehyde dehydrogenation, GTN-triggered enzyme inactivation, GTN denitration, NO formation, and soluble guanylate cyclase activation. Lack of the cysteines did not affect dehydrogenase activity but impeded GTN denitration, aggravated GTN-induced enzyme inactivation, and increased NO formation. A triple mutant lacking the cysteines and Glu268 catalyzed sustained formation of superstoichiometric amounts of NO and exhibited slower rates of inactivation. These results suggest three alternative pathways for the reaction of ALDH2 with GTN, all involving formation of a thionitrate/sulfenyl nitrite intermediate at Cys302 as the initial step. In the first pathway, which predominates in the wild-type enzyme and reflects clearance-based GTN denitration, the thionitrate apparently reacts with one of the adjacent cysteine residues to yield nitrite and a protein disulfide. The predominant reaction catalyzed by the single and double cysteine mutants requires Glu268 and results in irreversible enzyme inactivation. Finally, combined lack of the cysteines and Glu268 shifts the reaction toward formation of the free NO radical, presumably through homolytic cleavage of the sulfenyl nitrite intermediate. Although the latter reaction accounts for less than 10% of total turnover of GTN metabolism catalyzed by wild-type ALDH2, it is most likely essential for vascular GTN bioactivation.

  16. 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...

  17. Comparative site-directed mutagenesis in the catalytic amino acid triad in calicivirus proteases.

    Science.gov (United States)

    Oka, Tomoichiro; Murakami, Kosuke; Wakita, Takaji; Katayama, Kazuhiko

    2011-02-01

    Calicivirus proteases cleave the viral precursor polyprotein encoded by open reading frame 1 (ORF1) into multiple intermediate and mature proteins. These proteases have conserved histidine (His), glutamic acid (Glu) or aspartic acid (Asp), and cysteine (Cys) residues that are thought to act as a catalytic triad (i.e. general base, acid and nucleophile, respectively). However, is the triad critical for processing the polyprotein? In the present study, we examined these amino acids in viruses representing the four major genera of Caliciviridae: Norwalk virus (NoV), Rabbit hemorrhagic disease virus (RHDV), Sapporo virus (SaV) and Feline calicivirus (FCV). Using single amino-acid substitutions, we found that an acidic amino acid (Glu or Asp), as well as the His and Cys in the putative catalytic triad, cannot be replaced by Ala for normal processing activity of the ORF1 polyprotein in vitro. Similarly, normal activity is not retained if the nucleophile Cys is replaced with Ser. These results showed the calicivirus protease is a Cys protease and the catalytic triad formation is important for protease activity. Our study is the first to directly compare the proteases of the four representative calicivirus genera. Interestingly, we found that RHDV and SaV proteases critically need the acidic residues during catalysis, whereas proteolytic cleavage occurs normally at several cleavage sites in the ORF1 polyprotein without a functional acid residue in the NoV and FCV proteases. Thus, the substrate recognition mechanism may be different between the SaV and RHDV proteases and the NoV and FCV proteases. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

  18. Site-directed mutagenesis of photoprotein mnemiopsin: implication of some conserved residues in bioluminescence properties.

    Science.gov (United States)

    Mahdavi, Atiyeh; Sajedi, Reza H; Hosseinkhani, Saman; Taghdir, Majid; Sariri, Reyhaneh

    2013-03-01

    Mnemiopsin is a Ca(2+)-binding photoprotein from Mnemiopsis leidyi that emits a flash of blue light upon reacting with coelenterazine and Ca(2+). The light emission is a result of an intramolecular oxidation reaction. Similar to the other Ca(2+)-binding photoproteins, mnemiopsin is composed of apophotoprotein (206 amino acid residues), the imidazopyrazine chromophore, coelenterazine, and molecular oxygen. The biochemical properties of this photoprotein have been recently characterized but so far there has been no individual study on the role of critical residues. In this study, we introduced some mutations in the mnemiopsin structure for investigation of the roles of some critical residues in the substrate binding cavity, and neighboring residues in the mechanism of the reaction and the bioluminescence properties of the photoprotein. Mutants of mnemiopsin were produced by substitution of residues M77, W101 and M151. Three mutants (W101F, W101Y and M151Y mutants) had significantly reduced luminescence activity and altered bioluminescent properties (such as decay rate, Ca(2+) sensitivity, etc.), whereas the fourth (M77H mutant) lost its luminescence activity completely. Our experimental and theoretical studies suggest that residue M77 probably has structural importance and participates in stabilization of active site residues, whereas residue M151 is one of the critical mechanistic residues in ctenophore photoproteins.

  19. Pentalenene Synthase: Analysis of Active Site Residues by Site-Directed Mutagenesis

    NARCIS (Netherlands)

    Seemann, M.; Zhai, G.; Kraker, de J.W.; Paschall, C.M.; Christianson, D.W.; Cane, D.E.

    2002-01-01

    Incubation of farnesyl diphosphate (1) with the W308F or W308F/H309F mutants of pentalenene synthase, an enzyme from Streptomyces UC5319, yielded pentalenene (2), accompanied by varying proportions of (+)-germacrene A (7) with relatively minor changes in kcat and kcat/Km. By contrast, single H309 mu

  20. Site-directed mutagenesis of cation coordinating residues in the gastric H,K-ATPase.

    Science.gov (United States)

    Rulli, S J; Louneva, N M; Skripnikova, E V; Rabon, E C

    2001-03-01

    Site-mutations were introduced into putative cation binding site 1 of the H,K-ATPase at glu-797, thr-825, and glu-938. The side chain oxygen of each was not essential but the mutations produced different activation and inhibition kinetics. Site mutations thr-825 (ala, leu) and glu-938 (ala, gln) modestly decreased the apparent affinity to K+, while glu-797 (gln) was equivalent to wild type. As expected of competitive inhibition, mutations of thr-825 and glu-938 that decreased the apparent affinity for K+ also increased the apparent affinity for SCH28080. This is consistent with the participation of thr-825 and glu-938 in a cation binding domain. The sidechain geometry, but not the sidechain charge of glu-797, is essential to ATPase function as the site mutant glu-797 (gly) inactivated the H,K-ATPase, while glu-797 (gln) was active but the apparent affinity to SCH 28080 was decreased by four-fold. Lys-793, a unique residue of the H,K-ATPase, was essential for ATPase function. Since this residue is adjacent to site 1, the result suggests that charge pairing between lys-793 and residues at or near this site may be essential to ATPase function.

  1. Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis

    Science.gov (United States)

    Cellobiose 2-epimerase from the thermophile Caldicellulosiruptor saccharolyticus (CsCE) catalyzes the isomerization of lactose into lactulose, a non-digestible disaccharide widely used in food and pharmaceutical industries. Semi-rational approaches were applied to enhance the thermostability of CsCE...

  2. 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 E

  3. Improvement of Yarrowia lipolytica lipase enantioselectivity by using mutagenesis targeted to the substrate binding site.

    Science.gov (United States)

    Bordes, F; Cambon, E; Dossat-Létisse, V; André, I; Croux, C; Nicaud, J M; Marty, A

    2009-07-06

    Lip2p lipase from Yarrowia lipolytica was shown to be an efficient catalyst for the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates in the pharmaceutical industry. Enantioselectivity of this lipase was improved by site-directed mutagenesis targeted to the substrate binding site. To guide mutagenesis experiments, the three-dimensional model of this lipase was built by homology modelling techniques by using the structures of lipases from Rhizomucor miehei and Thermomyces lanuginosa as templates. On the basis of this structural model, five amino acid residues (T88, V94, D97, V232, V285) that form the hydrophobic substrate binding site of the lipase were selected for site-directed mutagenesis. Position 232 was identified as crucial for the discrimination between enantiomers. Variant V232A displayed an enantioselectivity enhanced by one order of magnitude, whereas variant V232L exhibited a selectivity inversion. To further explore the diversity, position 232 was systematically replaced by the 19 possible amino acids. Screening of this library led to the identification of the V232S variant, which has a tremendously increased E value compared to the parental enzyme for the resolution of 2-bromo-phenylacetic acid ethyl ester (58-fold) and 2-bromo-o-tolylacetic acid ethyl ester (16-fold). In addition to the gain in enantioselectivity, a remarkable increase in velocity was observed (eightfold increase) for both substrates.

  4. Sleeping Beauty transposon insertional mutagenesis based mouse models for cancer gene discovery

    Science.gov (United States)

    Moriarity, Branden S; Largaespada, David A

    2016-01-01

    Large-scale genomic efforts to study human cancer, such as the cancer gene atlas (TCGA), have identified numerous cancer drivers in a wide variety of tumor types. However, there are limitations to this approach, the mutations and expression or copy number changes that are identified are not always clearly functionally relevant, and only annotated genes and genetic elements are thoroughly queried. The use of complimentary, nonbiased, functional approaches to identify drivers of cancer development and progression is ideal to maximize the rate at which cancer discoveries are achieved. One such approach that has been successful is the use of the Sleeping Beauty (SB) transposon-based mutagenesis system in mice. This system uses a conditionally expressed transposase and mutagenic transposon allele to target mutagenesis to somatic cells of a given tissue in mice to cause random mutations leading to tumor development. Analysis of tumors for transposon common insertion sites (CIS) identifies candidate cancer genes specific to that tumor type. While similar screens have been performed in mice with the PiggyBac (PB) transposon and viral approaches, we limit extensive discussion to SB. Here we discuss the basic structure of these screens, screens that have been performed, methods used to identify CIS. PMID:26051241

  5. Combination Primer Polymerase Chain Reaction for Multi-Site Mutagenesis of Close Proximity Sites

    OpenAIRE

    Jensen, Pia Hønnerup; Weilguny, Dietmar

    2005-01-01

    We describe a rapid and efficient polymerase chain reaction procedure for multi-site-directed mutagenesis for cases in which the sites to be mutated are in close proximity. The combination primer polymer chain reaction method is based on a multi-site directed mutagenesis protocol together with a splicing by overlapping extension polymerase chain reaction protocol. several different combinations of multiple mutations were successfully performed with this method and are reported in this study.

  6. Identification of a rat model for usher syndrome type 1B by N-ethyl-N-nitrosourea mutagenesis-driven forward genetics.

    NARCIS (Netherlands)

    Smits, B.M.; Peters, T.A.; Mul, J.D.; Croes, H.J.E.; Fransen, J.A.M.; Beynon, A.J.; Guryev, V.; Plasterk, R.H.; Cuppen, E.P.J.G.

    2005-01-01

    The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis t

  7. Identification of a rat model for usher syndrome type 1B by N-ethyl-N-nitrosourea mutagenesis-driven forward genetics

    NARCIS (Netherlands)

    Smits, B.M.; Peters, T.A.; Mul, J.D.; Croes, H.J.; Fransen, J.A.; Beynon, A.J.; Guryev, V.; Plasterk, R.; Cuppen, E.

    2005-01-01

    The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis t

  8. Identification of a rat model for usher syndrome type 1B by N-ethyl-N-nitrosourea mutagenesis-driven forward genetics

    NARCIS (Netherlands)

    Smits, Bart M G; Peters, Theo A; Mul, Joram D; Croes, Huib J; Fransen, Jack A M; Beynon, Andy J; Guryev, Victor; Plasterk, Ronald H A; Cuppen, Edwin

    2005-01-01

    The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis t

  9. Identification of a rat model for usher syndrome type 1B by N-ethyl-N-nitrosourea mutagenesis-driven forward genetics.

    NARCIS (Netherlands)

    Smits, B.M.; Peters, T.A.; Mul, J.D.; Croes, H.J.E.; Fransen, J.A.M.; Beynon, A.J.; Guryev, V.; Plasterk, R.H.; Cuppen, E.P.J.G.

    2005-01-01

    The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis t

  10. 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.

  11. 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.

  12. Modeling of Anopheles minimus Mosquito NADPH-Cytochrome P450 Oxidoreductase (CYPOR and Mutagenesis Analysis

    Directory of Open Access Journals (Sweden)

    Pornpimol Rongnoparut

    2013-01-01

    Full Text Available Malaria is one of the most dangerous mosquito-borne diseases in many tropical countries, including Thailand. Studies in a deltamethrin resistant strain of Anopheles minimus mosquito, suggest cytochrome P450 enzymes contribute to the detoxification of pyrethroid insecticides. Purified A. minimus CYPOR enzyme (AnCYPOR, which is the redox partner of cytochrome P450s, loses flavin-adenosine di-nucleotide (FAD and FLAVIN mono-nucleotide (FMN cofactors that affect its enzyme activity. Replacement of leucine residues at positions 86 and 219 with phenylalanines in FMN binding domain increases FMN binding, enzyme stability, and cytochrome c reduction activity. Membrane-Bound L86F/L219F-AnCYPOR increases A. minimus P450-mediated pyrethroid metabolism in vitro. In this study, we constructed a comparative model structure of AnCYPOR using a rat CYPOR structure as a template. Overall model structure is similar to rat CYPOR, with some prominent differences. Based on primary sequence and structural analysis of rat and A. minimus CYPOR, C427R, W678A, and W678H mutations were generated together with L86F/L219F resulting in three soluble Δ55 triple mutants. The C427R triple AnCYPOR mutant retained a higher amount of FAD binding and increased cytochrome c reduction activity compared to wild-type and L86F/L219F-Δ55AnCYPOR double mutant. However W678A and W678H mutations did not increase FAD and NAD(PH bindings. The L86F/L219F double and C427R triple membrane-bound AnCYPOR mutants supported benzyloxyresorufin O-deakylation (BROD mediated by mosquito CYP6AA3 with a two- to three-fold increase in efficiency over wild-type AnCYPOR. The use of rat CYPOR in place of AnCYPOR most efficiently supported CYP6AA3-mediated BROD compared to all AnCYPORs.

  13. Large-scale screening of disease model through ENU mutagenesis in mice

    Institute of Scientific and Technical Information of China (English)

    HE Fang; WANG Zixing; ZHAO Jing; BAO Jie; DING Jun; RUAN Haibin; XIE Qing; ZHANG Zuoming; GAO Xiang

    2003-01-01

    Manipulation of mouse genome has merged as one of the most important approaches for studying gene function and establishing the disease model because of the high homology between human genome and mouse genome. In this study, the chemical mutagen ethylnitrosourea (ENU) was employed for inducing germ cell mutations in male C57BL/6J mice. The first generation (G1) of the backcross of these mutated mice, totally 3172, was screened for abnormal phenotypes on gross morphology, behavior, learning and memory, auditory brainstem response (ABR), electrocardiogram (ECG), electroretinogram (ERG), flash-visual evoked potential (F-VEP), bone mineral density, and blood sugar level. 595 mice have been identified with specific dominant abnormalities. Fur color changes, eye defects and hearing loss occurred at the highest frequency. Abnormalities related to metabolism alteration are least frequent. Interestingly, eye defects displayed significant left-right asymmetry and sex preference. Sex preference is also observed in mice with abnormal bone mineral density. Among 104 G1 generation mutant mice examined for inheritability, 14 of them have been confirmed for passing abnormal phenotypes to their progenies. However, we did not observe behavior abnormalities of G1 mice to be inheritable, suggesting multi-gene control for these complicated functions in mice. In conclusion, the generation of these mutants paves the way for understanding molecular and cellular mechanisms of these abnormal phenotypes, and accelerates the cloning of disease-related genes.

  14. 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

  15. 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.

  16. Transformation of MCF-10A cells by random mutagenesis with frameshift mutagen ICR191: A model for identifying candidate breast-tumor suppressors

    Directory of Open Access Journals (Sweden)

    Matsui Sei-Ichi

    2008-06-01

    Full Text Available Abstract Background Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of neoplastic transformation. Identifying frequent mutations in cancer cells suggests the involvement of mutant genes in carcinogenesis. Results To develop an in vitro model for the analysis of genetic alterations associated with breast carcinogenesis, we used random mutagenesis and selection of human non-tumorigenic immortalized breast epithelial cells MCF-10A in tissue-culture conditions that mimic tumor environment. Random mutations were generated in MCF-10A cells by cultivating them in a tissue-culture medium containing the frameshift-inducing agent ICR191. The first selective condition we used to transform MCF1-10A cells was cultivation in a medium containing mutagen at a concentration that allowed cell replication despite p53 protein accumulation induced by mutagen treatment. The second step of selection was either cell cultivation in a medium with reduced growth-factor supply or in a medium that mimics a hypoxia condition or growing in soft agar. Using mutagenesis and selection, we have generated several independently derived cultures with various degrees of transformation. Gene Identification by Nonsense-mediated mRNA decay Inhibition (GINI analysis has identified the ICR191-induced frameshift mutations in the TP53, smoothelin, Ras association (RalGDS/AF-6 domain family 6 (RASSF6 and other genes in the transformed MCF-10A cells. The TP53 gene mutations resulting in the loss of protein expression had been found in all independently transformed MCF-10A cultures, which form large progressively growing tumors with sustained angiogenesis in nude mice. Conclusion Identifying genes containing bi-allelic ICR191-induced frameshift mutations in the transformed MCF-10A cells generated by random mutagenesis and selection indicates putative breast-tumor suppressors. This

  17. 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.

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

    NARCIS (Netherlands)

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

    2006-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  20. Site-directed mutagenesis study of the three catalic residues of the fructosyltransferases of Lactobacillus reuteri 121.

    NARCIS (Netherlands)

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

    2004-01-01

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

  1. Site-Directed Mutagenesis of the Catalytic Residues Asp-52 and Glu-35 of Chicken Egg White Lysozyme

    National Research Council Canada - National Science Library

    Bruce A. Malcolm; Steven Rosenberg; Michael J. Corey; Judith S. Allen; Annie De Baetselier; Jack F. Kirsch

    1989-01-01

    ... (denoted as D52N and E35Q, respectively). The mutant enzyme D52N exhibits ≈ 5% of the wild-type lytic activity against Micrococcus luteus cell walls, while there is no measurable activity associated with E35Q (0.1% ± 0.1...

  2. Site-directed mutagenesis of the Anabaena sp. strain PCC 7120 nitrogenase active site to increase photobiological hydrogen production.

    Science.gov (United States)

    Masukawa, Hajime; Inoue, Kazuhito; Sakurai, Hidehiro; Wolk, C Peter; Hausinger, Robert P

    2010-10-01

    Cyanobacteria use sunlight and water to produce hydrogen gas (H₂), which is potentially useful as a clean and renewable biofuel. Photobiological H₂ arises primarily as an inevitable by-product of N₂ fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H₂ production by this strain, six nitrogenase amino acid residues predicted to reside within 5 Å of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N₂. Most of the 49 variants examined were deficient in N₂-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N₂ atmosphere significantly increased their in vivo rates of H₂ production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H₂ compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H₂ in an aerobic, nitrogen-containing environment.

  3. 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),

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

    National Research Council Canada - National Science Library

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

    2016-01-01

    .... HTP-OligoDesigner was created to provide the scientific community with a simple and intuitive online primer design tool for both laboratory-scale and high-throughput projects of sequence-independent...

  5. 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.

  6. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

    Science.gov (United States)

    Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

  7. [Stress-induced cellular adaptive mutagenesis].

    Science.gov (United States)

    Zhu, Linjiang; Li, Qi

    2014-04-01

    The adaptive mutations exist widely in the evolution of cells, such as antibiotic resistance mutations of pathogenic bacteria, adaptive evolution of industrial strains, and cancerization of human somatic cells. However, how these adaptive mutations are generated is still controversial. Based on the mutational analysis models under the nonlethal selection conditions, stress-induced cellular adaptive mutagenesis is proposed as a new evolutionary viewpoint. The hypothetic pathway of stress-induced mutagenesis involves several intracellular physiological responses, including DNA damages caused by accumulation of intracellular toxic chemicals, limitation of DNA MMR (mismatch repair) activity, upregulation of general stress response and activation of SOS response. These responses directly affect the accuracy of DNA replication from a high-fidelity manner to an error-prone one. The state changes of cell physiology significantly increase intracellular mutation rate and recombination activity. In addition, gene transcription under stress condition increases the instability of genome in response to DNA damage, resulting in transcription-associated DNA mutagenesis. In this review, we summarize these two molecular mechanisms of stress-induced mutagenesis and transcription-associated DNA mutagenesis to help better understand the mechanisms of adaptive mutagenesis.

  8. Spontaneous recurrent mutations and a complex rearrangement in the MECP2 gene in the light of current models of mutagenesis.

    Science.gov (United States)

    Todorov, Tihomir; Todorova, Albena; Motoescu, Cristina; Dimova, Petia; Iancu, Daniela; Craiu, Dana; Stoian, Daniela; Barbarii, Ligia; Bojinova, Veneta; Mitev, Vanyo

    2012-06-01

    Mutations in the methyl-CpG-binding protein 2 (MECP2) gene are associated with Rett syndrome (RTT). The MECP2 gene has some unique characteristics: (1) it is mainly affected by de novo mutations, due to recurrent independent mutational events in a defined "hot spot" regions or positions; (2) complex mutational events along a single allele are frequently found in this gene; (3) most mutations arise on paternal X chromosome. The recurrent point mutations involve mainly CpG dinucleotides, where C>T transitions are explained by methylation-mediated deamination. The complex mutational events might be explained by the genomic architecture of the region involving the MECP2 gene. The finding that most spontaneous mutations arise on paternal X-chromosome supports the higher contribution of replication-mediated mechanism of mutagenesis. We present 9 types of mutations in the MECP2 gene, detected in a group of 22 Bulgarian and 6 Romanian classical RTT patients. Thirteen patients were clarified on molecular level (46.4%). The point mutations in our sample account for 61.5%. One intraexonic deletion was detected in the present study (7.7%). One novel insertion c.321_322insGAAG, p.(Lys107_Leu108insGluAlafs2*) was found (7.7%). Large deletions and complex mutations account for 23%. A novel complex mutational event c.[584_624del41insTT; 638delTinsCA] was detected in a Romanian patient. We discuss different types of the MECP2 mutations detected in our sample in the light of the possible mechanisms of mutagenesis. Complex gene rearrangements involving a combination of deletions and insertions have always been most difficult to detect, to specify precisely and hence to explain in terms of their underlying mutational mechanisms.

  9. 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.

  10. Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf(-/-) mouse model.

    Science.gov (United States)

    Chapeau, Emilie A; Gembarska, Agnieszka; Durand, Eric Y; Mandon, Emeline; Estadieu, Claire; Romanet, Vincent; Wiesmann, Marion; Tiedt, Ralph; Lehar, Joseph; de Weck, Antoine; Rad, Roland; Barys, Louise; Jeay, Sebastien; Ferretti, Stephane; Kauffmann, Audrey; Sutter, Esther; Grevot, Armelle; Moulin, Pierre; Murakami, Masato; Sellers, William R; Hofmann, Francesco; Jensen, Michael Rugaard

    2017-03-21

    Inhibitors of double minute 2 protein (MDM2)-tumor protein 53 (TP53) interaction are predicted to be effective in tumors in which the TP53 gene is wild type, by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF, activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we used piggyBac (PB) transposon insertional mutagenesis to anticipate resistance mechanisms occurring during treatment with the MDM2-TP53 inhibitor HDM201. Constitutive PB mutagenesis in Arf(-/-) mice provided a collection of spontaneous tumors with characterized insertional genetic landscapes. Tumors were allografted in large cohorts of mice to assess the pharmacologic effects of HDM201. Sixteen out of 21 allograft models were sensitive to HDM201 but ultimately relapsed under treatment. A comparison of tumors with acquired resistance to HDM201 and untreated tumors identified 87 genes that were differentially and significantly targeted by the PB transposon. Resistant tumors displayed a complex clonality pattern suggesting the emergence of several resistant subclones. Among the most frequent alterations conferring resistance, we observed somatic and insertional loss-of-function mutations in transformation-related protein 53 (Trp53) in 54% of tumors and transposon-mediated gain-of-function alterations in B-cell lymphoma-extra large (Bcl-xL), Mdm4, and two TP53 family members, resulting in expression of the TP53 dominant negative truncations ΔNTrp63 and ΔNTrp73. Enhanced BCL-xL and MDM4 protein expression was confirmed in resistant tumors, as well as in HDM201-resistant patient-derived tumor xenografts. Interestingly, concomitant inhibition of MDM2 and BCL-xL demonstrated significant synergy in p53 wild-type cell lines in vitro. Collectively, our findings identify several potential mechanisms by which TP53 wild-type tumors may escape MDM2-targeted therapy.

  11. OneClick: A Program for Designing Focused Mutagenesis Experiments

    Directory of Open Access Journals (Sweden)

    Mark Warburton

    2015-07-01

    Full Text Available OneClick is a user-friendly web-based program, developed specifically for quick-and-easy design of focused mutagenesis experiments (e.g., site-directed mutagenesis and saturation mutagenesis. Written in Perl and developed into a web application using CGI programming, OneClick offers a step-by-step experimental design, from mutagenic primer design to analysis of a mutant library. Upon input of a DNA sequence encoding the protein of interest, OneClick designs the mutagenic primers according to user input, e.g., amino acid position to mutate, type of amino acid substitutions (e.g., substitution to a group of amino acids with similar chemical property and type of mutagenic primers. OneClick has incorporated an extensive range of commercially available plasmids and DNA polymerases suitable for focused mutagenesis. Therefore, OneClick also provides information on PCR mixture preparation, thermal cycling condition, expected size of PCR product and agar plate to use during bacterial transformation. Importantly, OneClick also carries out a statistical analysis of the resultant mutant library, information of which is important for selection/screening. OneClick is a unique and invaluable tool in the field of protein engineering, allowing for systematic construction of a mutant library or a protein variant and simplifying molecular biology work. The program will be constantly updated to reflect the rapid development in the fields of molecular biology and protein engineering.

  12. A combinatorial mutagenesis approach for functional epitope mapping on phage-displayed target antigen: application to antibodies against epidermal growth factor.

    Science.gov (United States)

    Infante, Yanelys Cabrera; Pupo, Amaury; Rojas, Gertrudis

    2014-01-01

    Although multiple different procedures to characterize the epitopes recognized by antibodies have been developed, site-directed mutagenesis remains the method of choice to define the energetic contribution of antigen residues to binding. These studies are useful to identify critical residues and to delineate functional maps of the epitopes. However, they tend to underestimate the roles of residues that are not critical for binding on their own, but contribute to the formation of the target epitope in an additive, or even cooperative, way. Mapping antigenic determinants with a diffuse energetic landscape, which establish multiple individually weak interactions with the antibody paratope, resulting in high affinity and specificity recognition of the epitope as a whole, is thus technically challenging. The current work was aimed at developing a combinatorial strategy to overcome the limitations of site-directed mutagenesis, relying on comprehensive randomization of discrete antigenic regions within phage-displayed antigen libraries. Two model antibodies recognizing epidermal growth factor were used to validate the mapping platform. Abrogation of antibody recognition due to the introduction of simultaneous replacements was able to show the involvement of particular amino acid clusters in epitope formation. The abundance of some of the original residues (or functionally equivalent amino acids sharing their physicochemical properties) among the set of mutated antigen variants selected on a given antibody highlighted their contributions and allowed delineation of a detailed functional map of the corresponding epitope. The use of the combinatorial approach could be expanded to map the interactions between other antigens/antibodies.

  13. 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

  14. 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

  15. Genome-wide ENU mutagenesis in combination with high density SNP analysis and exome sequencing provides rapid identification of novel mouse models of developmental disease.

    Directory of Open Access Journals (Sweden)

    Georgina Caruana

    Full Text Available BACKGROUND: Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU. METHODOLOGY/PRINCIPAL FINDINGS: ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS we identified novel recessive alleles for Fras1, Ift140 and Lig1. CONCLUSIONS/SIGNIFICANCE: In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.

  16. Examining the conformational dynamics of membrane proteins in situ with site-directed fluorescence labeling.

    Science.gov (United States)

    Richards, Ryan; Dempski, Robert E

    2011-05-29

    Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels, ion pumps, and transporters. Recent developments have combined site-specific fluorophore labeling alongside TEVC to concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells. We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling. Furthermore, this method provides an approach to determine distance constraints between specific residues. This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest. In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins. Finally, recent developments have also enabled the manipulation of select internal ions following co-expression with a second protein. Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (<5%) upon a conformational change of the protein. Second, these changes in fluorescence

  17. Generation of gene knockouts and mutant models in the laboratory rat by ENU-driven target-selected mutagenesis

    NARCIS (Netherlands)

    Smits, Bart M G; Mudde, Josine B; van de Belt, Jose; Verheul, Mark; Olivier, Jocelien; Homberg, Judith; Guryev, Victor; Cools, Alexander R; Ellenbroek, Bart A; Plasterk, Ronald H A; Cuppen, Edwin

    2006-01-01

    OBJECTIVE: The rat is one of the most important model organisms for biomedical and pharmacological research. However, the generation of novel models for studying specific aspects of human diseases largely depends on selection for specific traits using existing rat strains, thereby solely depending o

  18. Generation of gene knockouts and mutant models in the laboratory rat by ENU-driven target-selected mutagenesis.

    NARCIS (Netherlands)

    Smits, B.M.; Mudde, J.B.; Belt, J. van de; Verheul, M.; Olivier, J.; Homberg, J.R.; Guryev, V.; Cools, A.R.; Ellenbroek, B.A.; Plasterk, R.H.; Cuppen, E.

    2006-01-01

    OBJECTIVE: The rat is one of the most important model organisms for biomedical and pharmacological research. However, the generation of novel models for studying specific aspects of human diseases largely depends on selection for specific traits using existing rat strains, thereby solely depending o

  19. 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.

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

    Science.gov (United States)

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

    2016-06-01

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

  1. Coherent site-directed transport in complex molecular networks: an effective Hamiltonian approach.

    Science.gov (United States)

    Weissman, Shira; Peskin, Uri

    2010-03-21

    Defining the conditions for coherent site-directed transport from an electron donor to a specific acceptor through tunneling barriers in a network of multiple donor/acceptors sites is an important step toward controlling electronic processes in molecular networks. The required analysis is most challenging since the entire network in essentially involved in coherent transport. In this work we introduce an efficient approach for formulating an effective donor/acceptor coupling in terms of the entire network parameters. The approach is based on implementation of Feshbach projection operators to map the entire network Hamiltonian onto a subspace defined by two specific donor and acceptor sites. This nonperturbative approach enables to define regimes of network parameters in which the effective donor-acceptor coupling is optimal. This is demonstrated numerically for simple models of molecular networks.

  2. Homology modeling three-dimensional structure of AnxB1 and reducing its immunogenicity by sequence-deleted mutagenesis

    Institute of Scientific and Technical Information of China (English)

    YAN; Hongli; SONG; Yunlong; LIU; Fan; HE; Yan; SUN; Shuhan

    2004-01-01

    AnxB1,a novel annexin previously isolated from Cysticercus cellulose,shows high thrombi affinity and anticoagulant activity in vivo.In order to investigate the relationship between structure and biological function,a predicted three-dimensional(3D)model of AnxB1 was generated by homology modeling.This model contains four homologous internal-domains and the Cα trace of domain Ⅰ,Ⅱ and IV shows high similarity.Based on the structure characterization,four sequence-deleted mutants were constructed and expressed as GST fusion proteins in E.coli.Two of the mutants,GST-M3 and GST-M4 reserved high anticoagulant activity(p<0.01 vs.GST).Furthermore,compared with the wild type GST-AnxB1,the immunogenicity of GST-M3 and GST-M4 was reduced significantly(p<0.01)and the molecular weight was lowered to 27 kD and 34 kD,respectively.These observations laid a solid foundation for further study on developing new thrombolytic agents with higher efficiency and lower side effect.

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

    Directory of Open Access Journals (Sweden)

    Liguang Wang

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

  4. Germ cell mutagenesis in lambdalacZ transgenic mice treated with ethylnitrosourea : comparison with specific-locus test

    NARCIS (Netherlands)

    Delft, J.H.M. van; Baan, R.A.

    1995-01-01

    Germ cell mutagenesis was studied in male λlacZ transgenic mice in such a way that the data can be compared with literature data for germ cell mutagenesis obtained with the specific-locus test. This comparison is of interest for validation of the transgenic mouse model. We studied mutagenesis induce

  5. 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.

  6. The yeast I-Sce I meganuclease induces site-directed chromosomal recombination in mammalian cells.

    Science.gov (United States)

    Choulika, A; Perrin, A; Dujon, B; Nicolas, J F

    1994-11-01

    Double-strand breaks in genomic DNA stimulate recombination. Until now it was not possible to induce in vivo site-directed double-strand breaks in a mammalian chromosomal target. In this article we describe the use of I-Sce I meganuclease, a very rare cutter yeast endonuclease, to induce site-directed double-strand breaks mediated recombination. The results demonstrate the potential of the I-Sce I system for chromosome manipulation in mammalian cells.

  7. Optogenetic mutagenesis in Caenorhabditis elegans

    Science.gov (United States)

    Noma, Kentaro; Jin, Yishi

    2015-01-01

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

  8. PCR-based site-specific mutagenesis of peptide antibiotics FALL-39 and its biologic activities

    Institute of Scientific and Technical Information of China (English)

    Yun-xia YANG; Yun FENG; Bo-yao WANG; Qi WU

    2004-01-01

    AIM: To construct PGEX-1λT-FALL-39 expression vector and its mutant vector, and study the relationship of function and structure. METHODS: A cDNA encoding mature FALL-39 was cloned from SPCA- 1 cell mRNA and the prokaryotic expression vector PGEX- 1λT-FALL-39 was constructed. Two kinds of polymerase chain reaction (PCR) for the site-direction mutagenesis were used to construct FALL-39 mutant expression vector, FALL-39-Lys-32 and FALL-39-Lys-24. Minimal effective concentration, minimal inhibitory concentration, and minimal bactericidal concentration were used to assay the antibacterial activities of these peptides. Effects of different solution on the antibacterial activity of FALL-39 and FALL-39-Lys-32 were observed by CFU determination. The hemolytic effects of these peptides were also examined on human red blood cells. RESULTS: Two site-specific mutants FALL-39-Lys-32 and FALL-39-Lys24 were obtained by PCR-induced mutagenesis. In comparison with two-step PCR which required two pairs of primers, one step PCR which required one pair of primers is a simple and efficient method for the PCR based site-specific mutagenesis. Using the prokaryotic expression system, the E coli-based products of recombinant FALL39 and its mutant peptides were also obtained. The antibacterial assay showed that FALL-39-Lys-32 and FALL-39-Lys24 were more potential in the antibacterial activity against E coli ML35p and Pseltdomonas aeruginosa ATCC27853 than that of FALL-39, and no increase in hemolysis was observed at the antibacterial concentrations. The antibacterial activity of FALL-39-Lys-32 against E coli was more potent than that of FALL-39 in NaCl-containing LB medium, while its activity was almost the same as FALL-39 in SO2-4 containing Medium E. CONCLUSION: PCR-based mutagensis is a useful model system for studying the structure and function relationship of antimicrobial peptides. Keeping α-helical conformation of FALL-39 and increasing net positive charge can increase the

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-04-01

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

  10. Site-directed fluorescence studies of a prokaryotic ClC antiporter.

    Science.gov (United States)

    Bell, Susan P; Curran, Patricia K; Choi, Sean; Mindell, Joseph A

    2006-06-06

    Channels and transporters of the ClC family serve a variety of physiological functions. Understanding of their gating and transport mechanisms remains incomplete, with disagreement over the extent of protein conformational change involved. Using site-directed fluorescence labeling, we probe ClC-ec1, a prokaryotic ClC, for transport-related structural rearrangements. We specifically label cysteines introduced at several positions in the R helix of ClC-ec1 with AlexaFluor 488, an environment-sensitive fluorophore, and demonstrate that the labeled mutants show H+/Cl- transport activity indistinguishable from that of the wild-type protein. At each position that we examined we observe fluorescence changes upon acidification over the same pH range that is known to activate transport. The fluorescence change is also sensitive to Cl- concentration; furthermore, the Cl- and H+ dependencies are coupled as would be expected if the fluorescence change reflected a conformational change required for transport. Together, the results suggest that the changes in fluorescence report protein conformational changes underlying the transport process. Labeled transporters mutated to remove a glutamate critical to proton-coupled chloride transport retain pH-dependent fluorescence changes, suggesting that multiple residues confer pH dependence on the transport mechanism. These results have implications for models of transport and gating in ClC channels and transporters.

  11. TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice.

    Science.gov (United States)

    Li, Ting; Liu, Bo; Chen, Chih Ying; Yang, Bing

    2016-05-20

    Over the last decades, much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology. The breakthrough has been made in recent years with the advent of sequence-specific endonucleases, especially zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) guided nucleases (e.g., Cas9). In higher eukaryotic organisms, site-directed mutagenesis usually can be achieved through non-homologous end-joining (NHEJ) repair to the DNA double-strand breaks (DSBs) caused by the exogenously applied nucleases. However, site-specific gene replacement or genuine genome editing through homologous recombination (HR) repair to DSBs remains a challenge. As a proof of concept gene replacement through TALEN-based HR in rice (Oryza sativa), we successfully produced double point mutations in rice acetolactate synthase gene (OsALS) and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations. After ballistic delivery into rice calli of TALEN construct and donor DNA, nine HR events with different genotypes of OsALS were obtained in T0 generation at the efficiency of 1.4%-6.3% from three experiments. The HR-mediated gene edits were heritable to the progeny of T1 generation. The edited T1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance. The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.

  12. Crystal structure and site-directed mutational analysis reveals key residues involved in Escherichia coli ZapA function.

    Science.gov (United States)

    Roach, Elyse J; Kimber, Matthew S; Khursigara, Cezar M

    2014-08-22

    FtsZ is an essential cell division protein in Escherichia coli, and its localization, filamentation, and bundling at the mid-cell are required for Z-ring stability. Once assembled, the Z-ring recruits a series of proteins that comprise the bacterial divisome. Zaps (FtsZ-associated proteins) stabilize the Z-ring by increasing lateral interactions between individual filaments, bundling FtsZ to provide a scaffold for divisome assembly. The x-ray crystallographic structure of E. coli ZapA was determined, identifying key structural differences from the existing ZapA structure from Pseudomonas aeruginosa, including a charged α-helix on the globular domains of the ZapA tetramer. Key helix residues in E. coli ZapA were modified using site-directed mutagenesis. These ZapA variants significantly decreased FtsZ bundling in protein sedimentation assays when compared with WT ZapA proteins. Electron micrographs of ZapA-bundled FtsZ filaments showed the modified ZapA variants altered the number of FtsZ filaments per bundle. These in vitro results were corroborated in vivo by expressing the ZapA variants in an E. coli ΔzapA strain. In vivo, ZapA variants that altered FtsZ bundling showed an elongated phenotype, indicating improper cell division. Our findings highlight the importance of key ZapA residues that influence the extent of FtsZ bundling and that ultimately affect Z-ring formation in dividing cells.

  13. Structure of the inhibitory region of troponin by site directed spin labeling electron paramagnetic resonance

    Science.gov (United States)

    Brown, Louise J.; Sale, Ken L.; Hills, Ron; Rouviere, Clement; Song, Likai; Zhang, Xiaojun; Fajer, Piotr G.

    2002-01-01

    Site-directed spin labeling EPR (SDSL-EPR) was used to determine the structure of the inhibitory region of TnI in the intact cardiac troponin ternary complex. Maeda and collaborators have modeled the inhibitory region of TnI (skeletal 96–112: the structural motif that communicates the Ca2+ signal to actin) as a kinked α-helix [Vassylyev, D., Takeda, S., Wakatsuki, S., Maeda, K. & Maeda, Y. (1998) Proc. Natl. Acad. Sci. USA 95, 4847–4852), whereas Trewhella and collaborators have proposed the same region to be a flexible β-hairpin [Tung, C. S., Wall, M. E., Gallagher, S. C. & Trewhella, J. (2000) Protein Sci. 9, 1312–1326]. To distinguish between the two models, residues 129–145 of cardiac TnI were mutated sequentially to cysteines and labeled with the extrinsic spin probe, MTSSL. Sequence-dependent solvent accessibility was measured as a change in power saturation of the spin probe in the presence of the relaxation agent. In the ternary complex, the 129–137 region followed a pattern characteristic of a regular 3.6 residues/turn α-helix. The following region, residues 138–145, showed no regular pattern in solvent accessibility. Measurements of 4 intradomain distances within the inhibitory sequence, using dipolar EPR, were consistent with an α-helical structure. The difference in side-chain mobility between the ternary (C⋅I⋅T) and binary (C⋅I) complexes revealed a region of interaction of TnT located at the N-terminal end of the inhibitory sequence, residues 130–135. The above findings for the troponin complex in solution do not support either of the computational models of the binary complex; however, they are in very good agreement with a preliminary report of the x-ray structure of the cardiac ternary complex [Takeda, S. Yamashita, A., Maeda, K. & Maeda, Y. (2002) Biophys. J. 82, 832]. PMID:12239350

  14. 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.

  15. 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.

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

    DEFF Research Database (Denmark)

    Madec, Edwige; Stensballe, Allan; Kjellström, Sven

    2003-01-01

    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......C signalling cascade or for coupling to other signalling pathways. This is the first structure-function analysis of a bacterial receptor-like kinase of the Hanks family....

  17. 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)

  18. Sequencing of the amylopullulanase (apu) gene of Thermoanaerobacter ethanolicus 39E, and identification of the active site by site-directed mutagenesis.

    Science.gov (United States)

    Mathupala, S P; Lowe, S E; Podkovyrov, S M; Zeikus, J G

    1993-08-01

    The complete nucleotide sequence of the gene encoding the dual active amylopullulanase of Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum) was determined. The structural gene (apu) contained a single open reading frame 4443 base pairs in length, corresponding to 1481 amino acids, with an estimated molecular weight of 162,780. Analysis of the deduced sequence of apu with sequences of alpha-amylases and alpha-1,6 debranching enzymes enabled the identification of four conserved regions putatively involved in substrate binding and in catalysis. The conserved regions were localized within a 2.9-kilobase pair gene fragment, which encoded a M(r) 100,000 protein that maintained the dual activities and thermostability of the native enzyme. The catalytic residues of amylopullulanase were tentatively identified by using hydrophobic cluster analysis for comparison of amino acid sequences of amylopullulanase and other amylolytic enzymes. Asp597, Glu626, and Asp703 were individually modified to their respective amide form, or the alternate acid form, and in all cases both alpha-amylase and pullulanase activities were lost, suggesting the possible involvement of 3 residues in a catalytic triad, and the presence of a putative single catalytic site within the enzyme. These findings substantiate amylopullulanase as a new type of amylosaccharidase.

  19. X-ray structures of recombinant yeast cytochrome c peroxidase and three heme-cleft mutants prepared by site-directed mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Wang, J.; Mauro, J.M.; Edwards, S.L.; Oatley, S.J.; Fishel, L.A.; Ashford, V.A.; Xuong, Nguyenhuu; Kraut, J. (Univ. of California at San Diego, La Jolla (USA))

    1990-08-07

    The 2.2-{angstrom} x-ray structure for CCP(MI), a plasmid-encoded form of Saccharomyces cerevisiae cytochrome c peroxidase (CCP) expressed in Escherichia coli has been solved, together with the structures of three specifically designed single-site heme-cleft mutants. The structure of CCP(MI) was solved by using molecular replacement methods, since its crystals grow differently from the crystals of CCP isolated from bakers' yeast used previously for structural solution. Small distal-side differences between CCP(MI) and bakers' yeast CCP are observed, presumably due to a strain-specific Thr-53 {yields} Ile substitution in CCP(MI). The observation of a vacant sixth coordination site in this structure differs from the results of solution resonance Raman studies, which predict hexacoordinated high-spin iron. The coordination behavior of this W51F mutant is apparently altered in the presence of a precipitating agent, 30% 2-methyl-2,4-pentanediol. A proximal Trp-191 {yields} Phe mutant that has substantially diminished enzyme activity and altered magnetic properties accommodates the substitution by allowing the side chain of Phe-191, together with the segment of backbone to which it is attached, to move toward the heme. This relatively large local perturbation is accompanied by numerous small adjustments resulting in a slight overall compression of the enzyme's proximal domain; however, the iron coordination sphere is essentially unchanged. This structures rules out a major alteration in protein conformation as a reason for the dramatically decreased activity of the W191F mutant. From the alteration of local structure that occurs in this mutant, coupled with the results of preliminary functional studies, the authors infer that Asp-235 exerts influence on the heme iron so as to keep its sixth coordination site vacant, and hence reactive with peroxide substrate, over a wide pH range.

  20. Novel intracellular GH10 xylanase from Cohnella laeviribosi HY-21: biocatalytic properties and alterations of substrate specificities by site-directed mutagenesis of Trp residues.

    Science.gov (United States)

    Kim, Do Young; Han, Mi Kyoung; Oh, Hyun-Woo; Bae, Kyung Sook; Jeong, Tae-Sook; Kim, Sung Uk; Shin, Dong-Ha; Kim, In-Ho; Rhee, Young Ha; Son, Kwang-Hee; Park, Ho-Yong

    2010-11-01

    The novel intracellular GH10 xylanase (iXylC) gene (1023-bp) of Cohnella laeviribosi HY-21 encoded a protein consisting of 340 amino acids with a deduced molecular mass of 39,330Da and a calculated pI of 5.81. The primary structure of iXylC was 70% identical to that of Geobacillus sp. GH10 enzyme (GenBank accession number: EDV78425). Xylanolytic activity of the His-tagged iXylC overproduced in Escherichiacoli BL21 was stimulated by 2.2-fold in the presence of 0.5% non-ionic detergents. iXylC produced a mixture of xylooligosaccharides (xylobiose to xylooctaose) from xylotriose and xylotetraose used as the hydrolytic substrate. In addition, it exhibited considerable cleavage activities for p-nitrophenylxylopyranoside (PNP-xylopyranoside) and PNP-cellobioside, indicating that iXylC is a unique GH10 enzyme. The hydrolytic activity (57.8IUmL(-1)) of iXylC toward PNP-xylopyranoside increased to 8.3-fold by W217A and W315A mutations, while mutations of W133A, W295A, and W303A abolished the hydrolytic activity of the enzyme.

  1. Site-directed mutagenesis implicates a threonine residue in TM6 in the subtype selectivities of UH-AH 37 and pirenzepine at muscarinic receptors.

    Science.gov (United States)

    Ellis, J; Seidenberg, M

    2000-08-01

    The structural basis for the selectivity of the antagonist UH-AH 37 at human muscarinic acetylcholine receptors was investigated by expressing mutant receptors in COS-7 cells. Previous studies have demonstrated that the interaction between UH-AH 37 and [(3)H]N-methylscopolamine in equilibrium assays is competitive and that the high affinity of UH-AH 37 for the M(5) subtype, compared to M(2), is due to an epitope in the sixth transmembrane domain (TM6) or the third outer loop of the receptor. By mutating each nonconserved residue in this region of M(2) and M(5) to its counterpart in the other receptor, we identified a threonine residue in the middle of TM6 uniquely responsible for the higher affinity of the M(5) receptor (M(1), M(3), and M(4) receptors also carry a threonine at that location and also have high affinity for UH-AH 37). The mutant receptor in which the corresponding alanine of the M(2) receptor was replaced by threonine, M(2)(401)ala --> thr, expressed enhanced affinity for pirenzepine as well as for UH-AH 37. The chick M(2) receptor, which expresses anomalously high affinity for pirenzepine, differs from its mammalian counterparts by the presence of a threonine at this position. Affinities of AF-DX 116 and 4-DAMP, as well as the allosteric potency of UH-AH 37, were not sensitive to the M(2)(401) ala --> thr mutation. Copyright 2000 S. Karger AG, Basel

  2. Use of the steroid derivative RPR 106541 in combination with site-directed mutagenesis for enhanced cytochrome P-450 3A4 structure/function analysis.

    Science.gov (United States)

    Stevens, J C; Domanski, T L; Harlow, G R; White, R B; Orton, E; Halpert, J R

    1999-08-01

    RPR 106541 (20R-16alpha,17alpha-[butylidenebis(oxy)]-6al pha, 9alpha-difluoro-11beta-hydroxy-17beta-(methylthio)androst a-4-en-3-one) is an airway-selective steroid developed for the treatment of asthma. Two metabolites produced by human liver microsomes were identified as R- and S-sulfoxide diastereomers based on liquid chromatography/mass spectrometry analysis, proton nuclear magnetic resonance, and cochromatography with standards. Sulfoxide formation was determined to be cytochrome P-450 (CYP) 3A4-dependent by correlation with CYP3A4-marker nifedipine oxidase activity, inhibition by cyclosporin A and troleandomycin, and inhibition of R- (70%) and S- (64%) sulfoxide formation by anti-3A antibody. Expressed CYP2C forms catalyzed RPR 106541 sulfoxidation; however, other phenotyping approaches failed to confirm the involvement of CYP2C forms in these reactions in human liver microsomes. Expressed CYP3A4 catalyzed the formation of the sulfoxide diastereomers in a 1:1 ratio, whereas CYP3A5 displayed stereoselectivity for formation of the S-diastereomer. The high rate of sulfoxidation by CYP3A4 and the blockage of oxidative metabolism at the electronically favored 6beta-position provided advantages for RPR 106541 over other substrates as an active site probe of CYP3A4. Therefore, oxidation of RPR 106541 by various CYP3A4 substrate recognition site (SRS) mutants was assessed. In SRS-4, A305V and F304A showed dramatically reduced rates of R-diastereomer formation (83 and 64% decreases, respectively), but S-diastereomer formation was affected to a lesser extent. A370V (SRS-5) showed decreased formation of the R-sulfoxide (52%) but increased formation of the S-diastereomer. In the SRS-2 region, the most dramatic change in sulfoxide ratios was observed for L210A. In conclusion, the structure of RPR 106541 imposes specific constraints on enzyme binding and activity and thus represents an improved CYP3A4 probe substrate.

  3. PCR Site-Directed Mutagenesis of Wilson Disease Gene in Vitro%PCR技术对Wilson病基因进行定点突变的研究

    Institute of Scientific and Technical Information of China (English)

    闫振文; 梁秀龄; 等

    2002-01-01

    [目的] 利用聚合酶链反应(PCR)技术对Wilson病(WD)基因进行体外定点突变的研究.[方法] 采用PCR定点突变技术,首先设计两对引物,将突变位点设计在引物上,通过重叠延伸法两次PCR扩增,扩增片段上含有所需要的突变位点,最后将扩增片段克隆入pRc/CMV载体中.[结果] DNA测序表明在预期位点已经发生突变,WD基因第778位密码子由精氨酸(Arg)残基突变为亮氨酸残基(Leu),用PCR定点突变技术成功构建Wilson病基因突变体.[结论] PCR技术诱导定点突变准确、高效.Wilson病基因突变体的构建成功,为进一步研究该突变位点导致Wilson病的发病机制和Wilson病蛋白的结构和功能的关系奠定了基础.

  4. 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

  5. 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...

  6. 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

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

    NARCIS (Netherlands)

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

    2000-01-01

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

  8. 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, i

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

    Science.gov (United States)

    Banerjee, Aparajita; Preiser, Alyssa L.

    2016-01-01

    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. PMID:27548482

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

    DEFF Research Database (Denmark)

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

    1996-01-01

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

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

    DEFF Research Database (Denmark)

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

    2003-01-01

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

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

    Science.gov (United States)

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

    2010-04-01

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

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

    Science.gov (United States)

    Spangfort, Michael D; Mirza, Osman; Ipsen, Henrik; Van Neerven, R J Joost; Gajhede, Michael; Larsen, Jørgen N

    2003-09-15

    Specific allergy vaccination is an efficient treatment for allergic disease; however, the development of safer vaccines would enable a more general use of the treatment. Determination of molecular structures of allergens and allergen-Ab complexes facilitates epitope mapping and enables a rational 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 is a critical amino acid also in a major human IgE-binding epitope. By solving the three-dimensional crystal structure of the Bet v 1 Glu(45)-Ser mutant, it was shown that the change in immunochemical activity is directly related to the Glu(45)-Ser substitution and not to long-range structural alterations or collapse of the Bet v 1 mutant tertiary structure.

  14. Genetic analysis of nifF and nifA and site-directed mutagenesis of nifE in Azotobacter vinelandii

    OpenAIRE

    Bennett, Lisa Tracy

    1989-01-01

    Nitrogenase-catalyzed nitrogen fixation is a biochemically and genetically complex process requiring the participation of a number of different nif (nitrogen fixation) gene products. The nifF (electron transport), nifA (nif gene regulation) and nifE (FeMo-cofactor biosynthesis) genes from Azotobacter vinelandii were genetically analyzed. The nucleotide sequence of the nifF gene, which encodes a flavodoxin, was determined. Specific mutation strains indicated that in A vineland...

  15. 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 ...

  16. 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 ...

  17. 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...

  18. 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...

  19. Site-Directed Chemical Mutations on Abzymes: Large Rate Accelerations in the Catalysis by Exchanging the Functionalized Small Nonprotein Components.

    Science.gov (United States)

    Ishikawa, Fumihiro; Shirahashi, Masato; Hayakawa, Hiroshi; Yamaguchi, Asako; Hirokawa, Takatsugu; Tsumuraya, Takeshi; Fujii, Ikuo

    2016-10-21

    Taking advantage of antibody molecules to generate tailor-made binding sites, we propose a new class of protein modifications, termed as "site-directed chemical mutation." In this modification, chemically synthesized catalytic components with a variety of steric and electronic properties can be noncovalently and nongenetically incorporated into specific sites in antibody molecules to induce enzymatic activity. Two catalytic antibodies, 25E2 and 27C1, possess antigen-combining sites which bind catalytic components and act as apoproteins in catalytic reactions. By simply exchanging these components, antibodies 25E2 and 27C1 can catalyze a wide range of chemical transformations including acyl-transfer, β-elimination, aldol, and decarboxylation reactions. Although both antibodies were generated with the same hapten, phosphonate diester 1, they showed different catalytic activity. When phenylacetic acid 4 was used as the catalytic component, 25E2 efficiently catalyzed the elimination reaction of β-haloketone 2, whereas 27C1 showed no catalytic activity. In this work, we focused on the β-elimination reaction and examined the site-directed chemical mutation of 27C1 to induce activity and elucidate the catalytic mechanism. Molecular models showed that the cationic guanidyl group of Arg(H52) in 27C1 makes a hydrogen bond with the P═O oxygen in the hapten. This suggested that during β-elimination, Arg(H52) of 27C1 would form a salt bridge with the carboxylate of 4, thus destroying reactivity. Therefore, we utilized site-directed chemical mutation to change the charge properties of the catalytic components. When amine components 7-10 were used, 27C1 efficiently catalyzed the β-elimination reaction. It is noteworthy that chemical mutation with secondary amine 8 provided extremely high activity, with a rate acceleration [(kcat/Km 2)/kuncat] of 1 000 000. This catalytic activity likely arises from the proximity effect, plus general-base catalysis associated the

  20. The Role of Aspartic Acid 143 in E. coli tRNA-Guanine Transglycosylase: Insights from Mutagenesis Studies and Computational Modeling

    Science.gov (United States)

    Todorov, Katherine Abold; Tan, Xiao-Jian; Nonekowski, Susanne T.; Garcia, George A.; Carlson, Heather A.

    2005-01-01

    tRNA guanine transglycosylase (TGT) is a tRNA-modifying enzyme which catalyzes the posttranscriptional exchange of guanine in position 34 of tRNAY,H,N,D with the modified base queuine in eukaryotes or its precursor, preQ1 base, in eubacteria. Thus, TGT must recognize the guanine in tRNA and the free base queuine or preQ1 to catalyze this exchange. The crystal structure of Zymomonas mobilis TGT with preQ1 bound suggests that a key aspartate is critically involved in substrate recognition. To explore this, a series of site-directed mutants of D143 in Escherichia coli TGT were made and characterized to investigate heterocyclic substrate recognition. Our data confirm that D143 has significant impact on KM of guanine; however, the trend in the KM data (D143A D143A > D143N > D143S > D143T appears to be directly related to the degree of hydrogen bonding available to guanine in the binding site. PMID:15951383

  1. Glyoxyl-disulfide agarose: a tailor-made support for site-directed rigidification of proteins.

    Science.gov (United States)

    Godoy, Cesar A; de las Rivas, Blanca; Grazú, Valeria; Montes, Tamara; Guisán, José Manuel; López-Gallego, Fernando

    2011-05-09

    A new strategy has been developed for site-directed immobilization/rigidification of genetically modified enzymes through multipoint covalent attachment on bifunctional disulfide-glyoxyl supports. Here the mechanism is described as a two-step immobilization/rigidification protocol where the enzyme is directly immobilized by thiol-disulfide exchange between the β-thiol of the single genetically introduced cysteine and the few disulfide groups presented on the support surface (3 μmol/g). Afterward, the enzyme is uniquely rigidified by multipoint covalent attachment (MCA) between the lysine residues in the vicinity of the introduced cysteine and the many glyoxyl groups (220 μmol/g) on the support surface. Both site-directed immobilization and rigidification have been possible only on these novel bifunctional supports. In fact, this technology has made possible to elucidate the protein regions where rigidification by MCA promoted higher protein stabilizations. Hence, rigidification of vicinity of position 333 from lipase 2 from Geobacillus thermocatenulatus (BTL2) promoted a stabilization factor of 33 regarding the unipunctual site-directed immobilized derivative. In the same context, rigidification of penicillin G acylase from E. coli (PGA) through position β201 resulted in a stabilization factor of 1069. Remarkably, when PGA was site-directed rigidified through that position, it presented a half-life time of 140 h under 60% (v/v) of dioxane and 4 °C, meaning a derivative eight times more stable than the PGA randomly immobilized on glyoxyl-disulfide agarose. Herein we have opened a new scenario to optimize the stabilization of proteins via multipoint covalent immobilization, which may represent a breakthrough in tailor-made tridimensional rigidification of proteins.

  2. Chronic inflammation as a promotor of mutagenesis in essential thrombocythemia, polycythemia vera and myelofibrosis. A human inflammation model for cancer development?

    Science.gov (United States)

    Hasselbalch, Hans Carl

    2013-02-01

    The Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are acquired stem cell neoplasms, in which a stem cell lesion induces an autonomous proliferative advantage. In addition to the JAK2V617 mutation several other mutations have been described. Recently chronic inflammation has been proposed as a trigger and driver of clonal evolution in MPNs. Herein, it is hypothesized that sustained inflammation may elicit the stem cell insult by inducing a state of chronic oxidative stress with elevated levels of reactive oxygen species (ROS) in the bone marrow, thereby creating a high-risk microenvironment for induction of mutations due to the persistent inflammation-induced oxidative damage to DNA in hematopoietic cells. Alterations in the epigenome induced by the chronic inflammatory drive may likely elicit a "epigenetic switch" promoting persistent inflammation. The perspectives of chronic inflammation as the driver of mutagenesis in MPNs are discussed, including early intervention with interferon-alpha2 and potent anti-inflammatory agents (e.g. JAK1-2 inhibitors, histone deacetylase inhibitors, DNA-hypomethylators and statins) to disrupt the self-perpetuating chronic inflammation state and accordingly eliminating a potential trigger of clonal evolution and disease progression with myelofibrotic and leukemic transformation.

  3. Expression, characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus.

    Science.gov (United States)

    Yang, Yufeng; Huang, Lei; Wang, Jufang; Xu, Zhinan

    2015-01-01

    An FAD-dependent glucose dehydrogenase (FAD-GDH) from Aspergillus terreus NIH2624 was expressed in Escherichia coli with a yield of 228±16U/L of culture. Co-expression with chaperones DnaK/DnaJ/GrpE and osmotic stress induced by simple carbon sources enhanced productivity significantly, improving the yield to 23883±563U/L after optimization. FAD-GDH was purified in two steps with the specific activity of 604U/mg. Using d-glucose as substrate, the optimal pH and temperature for FAD-GDH were determined to be 7.5 and 50°C, respectively. Activity was stable across the pH range 3.5-9.0, and the half-life was 52min at 42°C. Km and Vmax were calculated as 86.7±5.3mM and 928±35U/mg, and the molecular weight was approximately 65.6kDa based on size exclusion chromatography, indicating a monomeric structure. The 3D structure of FAD-GDH was simulated by homology modelling using the structure of A. niger glucose oxidase (GOD) as template. From the model, His551, His508, Asn506 and Arg504 were identified as key residues, and their importance was verified by site-directed mutagenesis. Furthermore, three additional mutants (Arg84Ala, Tyr340Phe and Tyr406Phe) were generated and all exhibited a higher degree of substrate specificity than the native enzyme. These results extend our understanding of the structure and function of FAD-GDH, and could assist potential commercial applications.

  4. Coupled mutagenesis screens and genetic mapping in zebrafish.

    Science.gov (United States)

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

    2003-01-01

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

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

    DEFF Research Database (Denmark)

    Nielsen, Ulrik Dam

    2006-01-01

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

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

    DEFF Research Database (Denmark)

    Nielsen, Ulrik Dam

    2006-01-01

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

  7. 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.

  8. Directed mutagenesis as a probe of photosystem II function

    Energy Technology Data Exchange (ETDEWEB)

    Bowlby, N.; Sithole, I.; McIntosh, L. (MSU-DOE Plant Research Laboratory, East Lansing, MI (USA)); James, J.; Babcock, G. (Michigan State Univ., East Lansing (USA))

    1990-05-01

    Several mutants of Synechocystis 6803 have been generated by site-directed mutagenesis of psbA and psbD in order to study the roles of the D1 and D2 proteins during H{sub 2}O oxidation. Of key importance to these studies is the analysis of PSII electron transfer components such as Y{sub Z}{sup +} and Y{sub D}{sup +}, and the identification of possible ligands to Mn and Ca. Detailed information can be gained through EPR spectroscopy, however, sensitivity is limited by the PSII concentration of the sample. A simple procedure for the purification of PSII from this cyanobacterium, in sufficient amounts for EPR studies, will be presented. Preliminary results from mutant analysis extend the characterization of the two tyrosine radicals and suggest an important structural role for Pro{sup 162} of D1 and Pro{sup 161} of D2 in the ability of the photosystem to form the radical species on the adjacent tyrosine residues Tyr{sup 161} and Tyr{sup 160} on D1 and D2 respectively.

  9. History of environmental and molecular mutagenesis.

    Science.gov (United States)

    Hoffmann, George R

    2004-01-01

    Environmental and Molecular Mutagenesis, the journal of the Environmental Mutagen Society (EMS), marks its 25th anniversary in 2004. The journal, originally called Environmental Mutagenesis, was established in 1979 with Seymour Abrahamson as its first editor. The development of the journal is closely linked to the evolution of the fields of mutation research and genetic toxicology. This perspective traces the founding of the journal and discusses its editorial history, growth, content, style, administration, and relationship to the EMS.

  10. 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.

  11. Mechanisms of mutagenesis in mammalian cells. Application to human thyroid tumours; Mecanismes de mutagenese dans les cellules de mammifere. Le modele des tumeurs thyroidiennes humaines

    Energy Technology Data Exchange (ETDEWEB)

    Sarasin, A.; Bounacer, A.; Lepage, F.; Guillermo Suarez, H. [Institut de Recherches Scientifiques sur le Cancer, 94 - Villejuif (France). Laboratoire de Genetique Moleculaire, UPR 42 CNRS; Schlumberger, M. [Institut Gustave Roussy, 94 - Villejuif (France). Service de Medecine Nucleaire

    1999-03-01

    Mutations are defined as stable and irreversible modifications of the normal genetic message due to small changes in the number or type of bases, or to large modifications of the genome such as deletions, insertions or chromosome rearrangements. These lesions are due to either polymerase errors during normal DNA replication or unrepaired DNA lesions, which will give rise to mutations through a mutagenic pathway. The molecular process leading to mutagenesis depends largely on the type of DNA lesions. Base modifications, such as 8-oxo-guanine or thymine glycol, both induced by ionizing radiations (IR), are readily replicated leading to direct mutations, usually base-pair substitutions. The 8-oxo-G gives rise predominantly to G to T transversions, the type of mutations found in ras or p53 gene from IR-induced tumors. Bulky adducts produced by chemical carcinogens or UV-irradiation are usually repaired by the nucleotide excision repair (NER) pathway which is able to detect structural distortion in the normal double-strand DNA backbone. These lesions represent a blockage to DNA and RNA polymerases as well as some signal for p53 accumulation in the damaged cell. In the absence of repair, these lesions could be eventually replicated owing to the induction of specific proteins at least in bacteria during the SOS process. The precise nature of the error-prone replication across an un-excised DNA lesion in the template is not fully understood in detailed biochemical terms, in mammalian cells. IR basically produce a very large number of DNA lesions from unique base modifications to single- or double-strand breaks and even complex DNA lesions due to the passage of very high energy particles or to a local re-emission of numerous radicals. The breakage of the double-helix is a difficult lesionto repair. Either it will result in cell death or, after an incorrect re-combinational pathway, it will induce frame-shifts, large deletions or chromosomal rearrangements. Most of the IR

  12. Mutagenesis and modeling of the peroxiredoxin (Prx) complex with the NMR structure of ATP-bound human sulfiredoxin implicate aspartate 187 of Prx I as the catalytic residue in ATP hydrolysis.

    Science.gov (United States)

    Lee, Duck-Yeon; Park, Sung Jun; Jeong, Woojin; Sung, Ho Jin; Oho, Taena; Wu, Xiongwu; Rhee, Sue Goo; Gruschus, James M

    2006-12-26

    The catalytic cysteine of certain members of the peroxiredoxin (Prx) family can be hyperoxidized to cysteinesulfinic acid during reduction of peroxides. Sulfiredoxin is responsible for the ATP-dependent reduction of cysteinesulfinic acid (SO2H) of hyperoxidized Prx. Here we report the NMR solution structure of human sulfiredoxin (hSrx), both with and without bound ATP, and we model the complex of ATP-bound hSrx with Prx. Binding ATP causes only small changes in the NMR structure of hSrx, and the bound ATP conformation is quite similar to that seen for the previously reported X-ray structure of the ADP-hSrx complex. Although hSrx binds ATP, it does not catalyze hydrolysis by itself and has no catalytic acid residue typical of most ATPase and kinase family proteins. For modeling the complex, the ATP-bound hSrx was docked to hyperoxidized Prx II using EMAP of CHARMM. In the model complex, Asn186 of Prx II (Asp187 of Prx I) is in contact with the hSrx-bound ATP beta- and gamma-phosphate groups. Asp187 of Prx I was mutated to alanine and asparagine, and binding and activity of the mutants with hSrx were compared to those of the wild type. For the D187N mutant, both binding and hydrolysis and reduction activities were comparable to those of the wild type, whereas for D187A, binding was unimpaired but ATP hydrolysis and reduction did not occur. The modeling and mutagenesis analyses strongly implicate Asp187 of Prx I as the catalytic residue responsible for ATP hydrolysis in the cysteinesulfinic acid reduction of Prx by hSrx.

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

    National Research Council Canada - National Science Library

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

    2014-01-01

    .... We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens...

  14. DNA repair and mutagenesis of singlestranded bacteriophages

    Energy Technology Data Exchange (ETDEWEB)

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

    1981-01-01

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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

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

    Science.gov (United States)

    Nagy, Ervin D.; Bennetzen, Jeffrey L.

    2008-01-01

    The Pc locus of sorghum (Sorghum bicolor) determines dominant sensitivity to a host-selective toxin produced by the fungal pathogen Periconia circinata. The Pc region was cloned by a map-based approach and found to contain three tandemly repeated genes with the structures of nucleotide binding site–leucine-rich repeat (NBS–LRR) disease resistance genes. Thirteen independent Pc-to-pc mutations were analyzed, and each was found to remove all or part of the central gene of the threesome. Hence, this central gene is Pc. Most Pc-to-pc mutations were associated with unequal recombination. Eight recombination events were localized to different sites in a 560-bp region within the ∼3.7-kb NBS–LRR genes. Because any unequal recombination located within the flanking NBS–LRR genes would have removed Pc, the clustering of cross-over events within a 560-bp segment indicates that a site-directed recombination process exists that specifically targets unequal events to generate LRR diversity in NBS–LRR loci. PMID:18719093

  20. Lipase active-site-directed anchoring of organometallics: metallopincer/protein hybrids.

    Science.gov (United States)

    Kruithof, Cornelis A; Casado, Miguel A; Guillena, Gabriela; Egmond, Maarten R; van der Kerk-van Hoof, Anca; Heck, Albert J R; Klein Gebbink, Robertus J M; van Koten, Gerard

    2005-11-18

    The work described herein presents a strategy for the regioselective introduction of organometallic complexes into the active site of the lipase cutinase. Nitrophenol phosphonate esters, well known for their lipase inhibitory activity, are used as anchor functionalities and were found to be ideal tools to develop a single-site-directed immobilization method. A small series of phosphonate esters, covalently attached to ECE "pincer"-type d8-metal complexes through a propyl tether (ECE=[C6H3(CH2E)(2)-2,6]-; E=NR2 or SR), were designed and synthesized. Cutinase was treated with these organometallic phosphonate esters and the new metal-complex/protein hybrids were identified as containing exactly one organometallic unit per protein. The organometallic proteins were purified by membrane dialysis and analyzed by ESI-mass spectrometry. The major advantages of this strategy are: 1) one transition metal can be introduced regioselectively and, hence, the metal environment can potentially be fine-tuned; 2) purification procedures are facile due to the use of pre-synthesized metal complexes; and, most importantly, 3) the covalent attachment of robust organometallic pincer complexes to an enzyme is achieved, which will prevent metal leaching from these hybrids. The approach presented herein can be regarded as a tool in the development of regio- and enantioselective catalyst as well as analytical probes for studying enzyme properties (e.g., structure) and, hence, is a "proof-of-principle design" study in enzyme chemistry.

  1. 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.

  2. A new method for multilayered, site-directed immobilization of antibody on polystyrene surface.

    Science.gov (United States)

    Feng, Bo; Wang, Caiyun; Xie, Xiaomei; Feng, Xi; Li, Yuqin; Cao, Zhijian

    2014-07-18

    Polystyrene is a common substrate material for protein adsorption in biosensors and bioassays. Here, we present a new method for multilayered, site-directed immobilization of antibody on polystyrene surface through the linkage of a genetically engineered ligand and the assembly of staphylococcal protein A (SPA) with immunoglobulin G (IgG). In this method, antibodies were stacked on polystyrene surface layer by layer in a potential three-dimensional way and exposed the analyte-binding sites well. Enzyme-linked immunosorbent assay (ELISA) revealed that the new method showed a 32-fold higher detection sensitivity compared with the conventional one. Pull-down assay and Western blot analysis further confirmed that it is different from the ones of monolayer adsorption according to the comparison of adsorption capacity. The differentiated introduction of functional ligands, which is the key of this method, might offer a unique idea as a way to interfere with the dynamic behavior of a protein complex during the process of adsorption.

  3. Site-directed topical lidocaine spray attenuates perioperative respiratory adverse events in children undergoing elective surgery.

    Science.gov (United States)

    Li, Li-Wei; He, Long; Ai, Yanqiu; Chu, Qinjun; Zhang, Wei

    2016-06-01

    Perioperative respiratory adverse events (PRAEs) are a major cause of morbidity and mortality associated with pediatric anesthesia. Topical lidocaine administration reduces risk of PRAE in children undergoing elective endotracheal intubation. However, definitive evidence of its efficacy remains elusive, due, in part, to the wide variability in the methodology for spraying topical lidocaine. In this randomized controlled double-blind clinical trial, we sought to evaluate the effect of site-directed topical airway lidocaine, sprayed directly onto supraglottic, glottis, and subglottic areas, on the incidence of PRAE. The study population consisted of 322 children (age range, 6 mo-12 y), who were scheduled for an elective surgical procedure under general anesthesia with endotracheal intubation. Patients were randomly assigned to receive topical spray of lidocaine (group L) or saline (group S) over the supraglottic, glottis and subglottic areas under direct vision before tracheal intubation. Incidence of PRAE and time to extubation was recorded. There were no statistically significant intergroup differences with regard to baseline demographics, patient characteristics, and surgical parameters. Group L was associated with a significantly lower incidence of PRAE as compared with group S (12.80% versus 38.13%, respectively; P 2%; P = 0.005), and oxygen desaturation lidocaine over supraglottic, glottis, and subglottic areas before tracheal intubation significantly reduced the incidence of PRAE and a prolongation of extubation time in children. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Site-directed mutation of arginine 282 to glutamate uncouples the movement of peptides and protons by the rabbit proton-peptide cotransporter PepT1.

    Science.gov (United States)

    Meredith, David

    2004-04-16

    A conserved positive residue in the seventh transmembrane domain of the mammalian proton-coupled di- and tripeptide transporter PepT1 has been shown by site-directed mutagenesis to be a key residue for protein function. Substitution of arginine 282 with a glutamate residue (R282E-PepT1) gave a protein at the plasma membrane of Xenopus laevis oocytes that was able to transport the non-hydrolyzable dipeptide [3H]d-Phe-l-Gln, although unlike the wild type, the rate of transport by R282E-PepT1 was independent of the extracellular pH level, and the substrate could not be accumulated above equilibrium. The binding affinity of the mutant transport protein was unchanged from the wild type. Thus, R282E-Pept1 appears to have been changed from a proton-driven to a facilitated transporter for peptides. In addition, peptide transport by R282E-PepT1 still induced depolarization as measured by microelectrode recordings of membrane potential. A more detailed study by two-electrode voltage clamping revealed that R282E-PepT1 behaved as a peptide-gated non-selective cation channel with the ion selectivity series lithium > sodium > N-methyl-d-glucamine at pH 7.4. There was also a proton conductance (comparing pH 7.4 and 8.4), and at pH 5.5 the predominant conductance was for potassium ions. Therefore, it can be concluded that changing arginine 282 to a glutamate not only uncouples the cotransport of protons and peptides of the wild-type PepT1 but also creates a peptide-gated cation channel in the protein.

  5. Scientific opinion addressing the safety assessment of plants developed using Zinc Finger Nuclease 3 and other Site-Directed Nucleases with similar function

    Directory of Open Access Journals (Sweden)

    EFSA Panel on Genetically Modified Organisms (GMO

    2012-10-01

    Full Text Available

    The European Commission requested that the EFSA Panel on Genetically Modified Organisms deliver a scientific opinion related to risk assessment of plants developed using the zinc finger nuclease 3 technique (ZFN-3 which allows the integration of gene(s in a predefined insertion site in the genome of the recipient species. Since other nucleases with a similar function to ZFN are considered in this opinion the term site-directed nuclease 3 (SDN-3 is used to describe the technique rather than ZFN-3 specifically. The EFSA GMO Panel considers that its guidance documents are applicable for the evaluation of food and feed products derived from plants developed using the SDN-3 technique and for performing an environmental risk assessment. However, on a case-by-case basis lesser amounts of event specific data may be needed for the risk assessment of plants developed using the SDN-3 technique. The EFSA GMO Panel compared the hazards associated with plants produced by the SDN-3 technique with those obtained by conventional plant breeding techniques and by currently used transgenesis. With respect to the genes introduced, the SDN-3 technique does not differ from transgenesis or from the other genetic modification techniques currently used, and can be used to introduce transgenes, intragenes or cisgenes. The main difference between the SDN-3 technique and transgenesis is that the insertion of DNA is targeted to a predefined region of the genome. Therefore, the SDN-3 technique can minimise hazards associated with the disruption of genes and/or regulatory elements in the recipient genome. Whilst the SDN-3 technique can induce off-target changes in the genome of the recipient plant these would be fewer than those occurring with most mutagenesis techniques. Furthermore, where such changes occur they would be of the same types as those produced by conventional breeding techniques.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sandra Witz

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

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

    KAUST Repository

    Witz, Sandra

    2014-03-12

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

  10. Atomic modelling and systematic mutagenesis identify residues in multiple drug binding sites that are essential for drug resistance in the major Candida transporter Cdr1.

    Science.gov (United States)

    Nim, Shweta; Lobato, Lucia Gonzalez; Moreno, Alexis; Chaptal, Vincent; Rawal, Manpreet Kaur; Falson, Pierre; Prasad, Rajendra

    2016-11-01

    The ABC (ATP-Binding Cassette) transporter Cdr1 (Candida drug resistance 1) protein (Cdr1p) of Candida albicans, shows promiscuity towards the substrate it exports and plays a major role in antifungal resistance. It has two transmembrane domains (TMDs) comprising of six transmembrane helices (TMH) that envisage and confer the substrate specificity and two nucleotide binding domains (NBDs), interconnected by extracellular loops (ECLs) and intracellular loops (ICLs) Cdr1p. This study explores the diverse substrate specificity spectrum to get a deeper insight into the structural and functional features of Cdr1p. By screening with the variety of compounds towards an in-house TMH 252 mutant library of Cdr1p, we establish new substrates of Cdr1p. The localization of substrate-susceptible mutants in an ABCG5/G8 homology model highlights the common and specific binding pockets inside the membrane domain, where rhodamines and tetrazoliums mainly engage the N-moiety of Cdr1p, binding between TMH 2, 11 and surrounded by TMH 1, 5. Whereas, tin chlorides involve both N and C moieties located at the interface of TMH 2, 11, 1 and 5. Further, screening of the in house TMH mutant library of Cdr1p displays the TMH12 interaction with tetrazolium chloride, trimethyltin chloride and a Ca(2+) ionophore, A23187. In silico localization reveals a binding site at the TMH 12, 9 and 10 interface, which is widely exposed to the lipid interface. Together, for the first time, our study shows the molecular localization of Cdr1p substrates-binding sites and demonstrates the participation of TMH12 in a peripheral drug binding site.

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

    Directory of Open Access Journals (Sweden)

    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.

  12. [Mutagenesis of the human histamine H1 receptor and design of new antihistamine agents].

    Science.gov (United States)

    Differding, E; Gillard, M; Moguilevsky, N; Varsalona, F; Noyer, M; Daliers, J; Goldstein, S; Neuwels, M; Lassoie, M A; Guillaume, J P; Bascour, M; Bollen, A; Hénichart, J P

    1996-01-01

    The binding cavity of histamine and histamine antagonists is explored using site directed mutagenesis of the human histamine H1 receptor and the amino acids involved in ligand binding are identified. Whereas Asp107 and Phe199 are important for both agonists and antagonists, two additional amino acids (Asn198 and Trp103) are required for efficient histamine binding. The binding site of antagonists is best defined as resulting from a strong ionic bond to Asp107, an orthogonal interaction between one of the aromatic rings with Phe199, and probably a hydrophobic interaction between the second aromatic ring and the lipophilic amino acids of the upper part of TMIV and TMV. This is consistent with structure-activity data of most described antagonists.

  13. X-Ray Structure and Mutagenesis Studies of the N-Isopropylammelide Isopropylaminohydrolase, AtzC.

    Directory of Open Access Journals (Sweden)

    Sahil Balotra

    Full Text Available The N-isopropylammelide isopropylaminohydrolase from Pseudomonas sp. strain ADP, AtzC, provides the third hydrolytic step in the mineralization of s-triazine herbicides, such as atrazine. We obtained the X-ray crystal structure of AtzC at 1.84 Å with a weak inhibitor bound in the active site and then used a combination of in silico docking and site-directed mutagenesis to understand the interactions between AtzC and its substrate, isopropylammelide. The substitution of an active site histidine residue (His249 for an alanine abolished the enzyme's catalytic activity. We propose a plausible catalytic mechanism, consistent with the biochemical and crystallographic data obtained that is similar to that found in carbonic anhydrase and other members of subtype III of the amidohydrolase family.

  14. Engineering cofactor preference of ketone reducing biocatalysts: A mutagenesis study on a γ-diketone reductase from the yeast Saccharomyces cerevisiae serving as an example.

    Science.gov (United States)

    Katzberg, Michael; Skorupa-Parachin, Nàdia; Gorwa-Grauslund, Marie-Françoise; Bertau, Martin

    2010-04-14

    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 gamma-diketone 2,5-hexanedione furnishing the diol (2S,5S)-hexanediol and the gamma-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.

  15. 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.

  16. Ligand-induced changes in estrogen receptor conformation as measured by site-directed spin labeling.

    Science.gov (United States)

    Hurth, Kyle M; Nilges, Mark J; Carlson, Kathryn E; Tamrazi, Anobel; Belford, R Linn; Katzenellenbogen, John A

    2004-02-24

    Site-directed spin labeling (SDSL), the site-specific incorporation of nitroxide spin-labels into a protein, has allowed us to investigate ligand-induced conformational changes in the ligand-binding domain of human estrogen receptor alpha (hERalpha-LBD). EPR (electron paramagnetic resonance) spectroscopy of the nitroxide probe attached to ER produces different spectra depending upon the identity of the bound ligand; these differences are indicative of changes in the type and degree of motional character of the spin-label induced by different ligand-induced conformations of labeled ER. Visual inspection of EPR spectra, construction of B versus C cross-correlation plots, and cross-comparison of spectral pairs using a relative squared difference (RSD) calculation allowed receptor-ligand complexes to be profiled according to their conformational character. Plotting B and C parameters allowed us to evaluate the liganded receptor according to the motional characteristics of the attached spin-label, and they were particularly illustrative for the receptor labeled at position 530, which had motion between the fast and intermediate regimes. RSD analysis allowed us to directly compare the similarity or difference between two different spectra, and these comparisons produced groupings that paralleled those seen in B versus C cross-correlation plots, again relating meaningfully with the pharmacological nature of the bound ligand. RSD analysis was also particularly useful for qualifying differences seen with the receptor labeled at position 417, which had motion between the intermediate and slow motional regimes. This work demonstrates that B and C formulas from EPR line shape theory are useful for qualitative analysis of spectra with differences subtler than those that are often analyzed by EPR spectroscopists. This work also provides evidence that the ER can exist in a range of conformations, with specific conformations resulting from preferential stabilization of ER by the

  17. 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

  18. A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing.

    Science.gov (United States)

    Stiffler, Michael A; Subramanian, Subu K; Salinas, Victor H; Ranganathan, Rama

    2016-07-03

    Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 β-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to β-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

  19. Characterization of Site-Directed Mutants in the lac Permease of Escherichia coli. 1. Replacement of Histidine Residues

    NARCIS (Netherlands)

    Püttner, Irene B.; Sarkar, Hemanta K.; Padan, Etana; Lolkema, Julius S.; Kaback, H. Ronald

    1989-01-01

    Wild-type lac permease from Escherichia coli and two site-directed mutant permeases containing Arg in place of His35 and His39 or His322 were purified and reconstituted into proteoliposomes. H35-39R permease is indistinguishable from wild type with regard to all modes of translocation. In contrast,

  20. A plasmid-coded and site-directed mutation in Escherichia coli 23S RNA that confers resistance to erythromycin

    DEFF Research Database (Denmark)

    Vester, Birte; Garrett, Roger Antony

    1987-01-01

    Primer-directed mutagenesis was employed to introduce an A2058----G transition in plasmid-encoded Escherichia coli 23S RNA at a site that has been implicated, indirectly, in erythromycin binding. The mutation raises the growth tolerance of cells from 30 to 300 micrograms/ml of erythromycin, and c...

  1. Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns.

    Science.gov (United States)

    Davenport, Kaitlynn R; Smith, Christopher A; Hofstetter, Heike; Horn, James R; Hofstetter, Oliver

    2016-05-15

    In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first

  2. A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.

    Science.gov (United States)

    Magnani, Francesca; Serrano-Vega, Maria J; Shibata, Yoko; Abdul-Hussein, Saba; Lebon, Guillaume; Miller-Gallacher, Jennifer; Singhal, Ankita; Strege, Annette; Thomas, Jennifer A; Tate, Christopher G

    2016-08-01

    The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes ∼6-12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of ∼300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an (125)I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies.

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

    Science.gov (United States)

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

    2012-02-15

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

  4. Novel Random Mutagenesis Method for Directed Evolution.

    Science.gov (United States)

    Feng, Hong; Wang, Hai-Yan; Zhao, Hong-Yan

    2017-01-01

    Directed evolution is a powerful strategy for gene mutagenesis, and has been used for protein engineering both in scientific research and in the biotechnology industry. The routine method for directed evolution was developed by Stemmer in 1994 (Stemmer, Proc Natl Acad Sci USA 91, 10747-10751, 1994; Stemmer, Nature 370, 389-391, 1994). Since then, various methods have been introduced, each of which has advantages and limitations depending upon the targeted genes and procedure. In this chapter, a novel alternative directed evolution method which combines mutagenesis PCR with dITP and fragmentation by endonuclease V is described. The kanamycin resistance gene is used as a reporter gene to verify the novel method for directed evolution. This method for directed evolution has been demonstrated to be efficient, reproducible, and easy to manipulate in practice.

  5. The mechanisms of UV mutagenesis.

    Science.gov (United States)

    Ikehata, Hironobu; Ono, Tetsuya

    2011-01-01

    Ultraviolet (UV) light induces specific mutations in the cellular and skin genome such as UV-signature and triplet mutations, the mechanism of which has been thought to involve translesion DNA synthesis (TLS) over UV-induced DNA base damage. Two models have been proposed: "error-free" bypass of deaminated cytosine-containing cyclobutane pyrimidine dimers (CPDs) by DNA polymerase η, and error-prone bypass of CPDs and other UV-induced photolesions by combinations of TLS and replicative DNA polymerases--the latter model has also been known as the two-step model, in which the cooperation of two (or more) DNA polymerases as misinserters and (mis)extenders is assumed. Daylight UV induces a characteristic UV-specific mutation, a UV-signature mutation occurring preferentially at methyl-CpG sites, which is also observed frequently after exposure to either UVB or UVA, but not to UVC. The wavelengths relevant to the mutation are so consistent with the composition of daylight UV that the mutation is called solar-UV signature, highlighting the importance of this type of mutation for creatures with the cytosine-methylated genome that are exposed to the sun in the natural environment. UVA has also been suggested to induce oxidative types of mutation, which would be caused by oxidative DNA damage produced through the oxidative stress after the irradiation. Indeed, UVA produces oxidative DNA damage not only in cells but also in skin, which, however, does not seem sufficient to induce mutations in the normal skin genome. In contrast, it has been demonstrated that UVA exclusively induces the solar-UV signature mutations in vivo through CPD formation.

  6. Whole-Genome Profiling of a Novel Mutagenesis Technique Using Proofreading-Deficient DNA Polymerase δ

    Directory of Open Access Journals (Sweden)

    Yuh Shiwa

    2012-01-01

    Full Text Available A novel mutagenesis technique using error-prone DNA polymerase δ (polδ, the disparity mutagenesis model of evolution, has been successfully employed to generate novel microorganism strains with desired traits. However, little else is known about the spectra of mutagenic effects caused by disparity mutagenesis. We evaluated and compared the performance of the polδMKII mutator, which expresses the proofreading-deficient and low-fidelity polδ, in Saccharomyces cerevisiae haploid strain with that of the commonly used chemical mutagen ethyl methanesulfonate (EMS. This mutator strain possesses exogenous mutant polδ supplied from a plasmid, tthereby leaving the genomic one intact. We measured the mutation rate achieved by each mutagen and performed high-throughput next generation sequencing to analyze the genome-wide mutation spectra produced by the 2 mutagenesis methods. The mutation frequency of the mutator was approximately 7 times higher than that of EMS. Our analysis confirmed the strong G/C to A/T transition bias of EMS, whereas we found that the mutator mainly produces transversions, giving rise to more diverse amino acid substitution patterns. Our present study demonstrated that the polδMKII mutator is a useful and efficient method for rapid strain improvement based on in vivo mutagenesis.

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

    Science.gov (United States)

    Varshney, Gaurav Kumar

    2014-01-01

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

  8. Amino Acid Hydrolysis and Analysis System for Investigation of Site Directed Nucleation and Growth of Ceramic Films on Metallic Surfaces

    Science.gov (United States)

    2008-09-30

    of biomineralization by hemocytes isolated from a bivalve on a metal substrate outside of the organism. These results indicate that it is possible...to deposit a ceramic material onto a metal substrate at ambient temperature and pressure. Figure I A-B. SEM evidence of cellular biomineralization on...hydrolysis and analysis system for investigation of site directed nucleation and growth of cera1nic films on metallic surfaces Sb. GRANT NUMBER

  9. Long-distance effects of insertional mutagenesis.

    Directory of Open Access Journals (Sweden)

    Ruchi Singhal

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Kaldor, J.

    1983-03-01

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

  11. An efficient TALEN mutagenesis system in rice.

    Science.gov (United States)

    Chen, Kunling; Shan, Qiwei; Gao, Caixia

    2014-08-15

    Targeted gene mutagenesis is a powerful tool for elucidating gene function and facilitating genetic improvement in rice. TALENs (transcription activator-like effector nucleases), consisting of a custom TALE DNA binding domain fused to a nonspecific FokI cleavage domain, are one of the most efficient genome engineering methods developed to date. The technology of TALENs allows DNA double-strand breaks (DSBs) to be introduced into predetermined chromosomal loci. DSBs trigger DNA repair mechanisms and can result in loss of gene function by error-prone non-homologous end joining (NHEJ), or they can be exploited to modify gene function or activity by precise homologous recombination (HR). In this paper, we describe a detailed protocol for constructing TALEN expression vectors, assessing nuclease activities in vivo using rice protoplast-based assays, generating and introducing TALEN DNAs into embryogenic calluses of rice and identifying TALEN-generated mutations at targeted genomic sites. Using these methods, T0 rice plants resulting from TALEN mutagenesis can be produced within 4-5 months.

  12. A disulfide intercalator toolbox for the site-directed modification of polypeptides.

    Science.gov (United States)

    Wang, Tao; Wu, Yuzhou; Kuan, Seah Ling; Dumele, Oliver; Lamla, Markus; Ng, David Y W; Arzt, Matthias; Thomas, Jessica; Mueller, Jan O; Barner-Kowollik, Christopher; Weil, Tanja

    2015-01-02

    A disulfide intercalator toolbox was developed for site-specific attachment of a broad variety of functional groups to proteins or peptides under mild, physiological conditions. The peptide hormone somatostatin (SST) served as model compound for intercalation into the available disulfide functionalization schemes starting from the intercalator or the reactive SST precursor before or after bioconjugation. A tetrazole-SST derivative was obtained that undergoes photoinduced cycloaddition in mammalian cells, which was monitored by live-cell imaging. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

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

    2004-01-01

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

  14. DNA abasic site-directed formation of fluorescent silver nanoclusters for selective nucleobase recognition

    Science.gov (United States)

    Ma, Kun; Cui, Qinghua; Liu, Guiying; Wu, Fei; Xu, Shujuan; Shao, Yong

    2011-07-01

    DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation related diseases. Various methods for SNP detection have been proposed and many are already in use. Here, we find that the abasic site (AP site) in the DNA duplex can be developed as a capping scaffold for the generation of fluorescent silver nanoclusters (Ag NCs). As a proof of concept, the DNA sequences from fragments near codon 177 of cancer supression gene p53 were used as a model for SNP detection by in situ formed Ag NCs. The formation of fluorescent Ag NCs in the AP site-containing DNA duplex is highly selective for cytosine facing the AP site and guanines flanking the site and can be employed in situ as readout for SNP detection. The fluorescent signal-on sensing for SNP based on this inorganic fluorophore is substantially advantageous over the previously reported signal-off responses using low-molecular-weight organic ligands. The strong dependence of fluorescent Ag NC formation on the sequences surrounding the AP site was successfully used to identify mutations in codon 177 of cancer supression gene p53. We anticipate that this approach will be employed to develop a practical SNP detection method by locating an AP site toward the midway cytosine in a target strand containing more than three consecutive cytosines.

  15. DNA abasic site-directed formation of fluorescent silver nanoclusters for selective nucleobase recognition

    Energy Technology Data Exchange (ETDEWEB)

    Ma Kun; Cui Qinghua; Liu Guiying; Wu Fei; Xu Shujuan; Shao Yong, E-mail: yshao@zjnu.cn [Institute of Physical Chemistry, Zhejiang Normal University, Jinhua 321004, Zhejiang (China)

    2011-07-29

    DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation related diseases. Various methods for SNP detection have been proposed and many are already in use. Here, we find that the abasic site (AP site) in the DNA duplex can be developed as a capping scaffold for the generation of fluorescent silver nanoclusters (Ag NCs). As a proof of concept, the DNA sequences from fragments near codon 177 of cancer supression gene p53 were used as a model for SNP detection by in situ formed Ag NCs. The formation of fluorescent Ag NCs in the AP site-containing DNA duplex is highly selective for cytosine facing the AP site and guanines flanking the site and can be employed in situ as readout for SNP detection. The fluorescent signal-on sensing for SNP based on this inorganic fluorophore is substantially advantageous over the previously reported signal-off responses using low-molecular-weight organic ligands. The strong dependence of fluorescent Ag NC formation on the sequences surrounding the AP site was successfully used to identify mutations in codon 177 of cancer supression gene p53. We anticipate that this approach will be employed to develop a practical SNP detection method by locating an AP site toward the midway cytosine in a target strand containing more than three consecutive cytosines.

  16. Scoring function to predict solubility mutagenesis

    Directory of Open Access Journals (Sweden)

    Deutsch Christopher

    2010-10-01

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

  17. Comparing Different Strategies in Directed Evolution of Enzyme Stereoselectivity: Single- versus Double-Code Saturation Mutagenesis.

    Science.gov (United States)

    Sun, Zhoutong; Lonsdale, Richard; Li, Guangyue; Reetz, Manfred T

    2016-10-04

    Saturation mutagenesis at sites lining the binding pockets of enzymes constitutes a viable protein engineering technique for enhancing or inverting stereoselectivity. Statistical analysis shows that oversampling in the screening step (the bottleneck) increases astronomically as the number of residues in the randomization site increases, which is the reason why reduced amino acid alphabets have been employed, in addition to splitting large sites into smaller ones. Limonene epoxide hydrolase (LEH) has previously served as the experimental platform in these methodological efforts, enabling comparisons between single-code saturation mutagenesis (SCSM) and triple-code saturation mutagenesis (TCSM); these employ either only one or three amino acids, respectively, as building blocks. In this study the comparative platform is extended by exploring the efficacy of double-code saturation mutagenesis (DCSM), in which the reduced amino acid alphabet consists of two members, chosen according to the principles of rational design on the basis of structural information. The hydrolytic desymmetrization of cyclohexene oxide is used as the model reaction, with formation of either (R,R)- or (S,S)-cyclohexane-1,2-diol. DCSM proves to be clearly superior to the likewise tested SCSM, affording both R,R- and S,S-selective mutants. These variants are also good catalysts in reactions of further substrates. Docking computations reveal the basis of enantioselectivity.

  18. Mutagenesis of bacteriophage IKe major coat protein transmembrane domain: role of an interfacial proline residue.

    Science.gov (United States)

    Williams, K A; Deber, C M

    1993-10-15

    The transmembrane (TM) domain of the 53-residue major coat protein of the M13-related bacteriophage IKe (residues 24-42: LISQTWPVVTTVVVAGVLI) has been subjected to randomized mutagenesis to probe the conformation and stability of the TM domain, as well as the effect of structurally-important residues such as proline. TM mutants were obtained by the Eckstein method of site-directed mutagenesis using the IKe genome as template so as to eliminate the need for subcloning. Over 40 single- and double-site viable mutants of bacteriophage IKe were isolated. Every residue in the TM segment, except the highly conserved Trp29, could be mutated to at least one other residue; polar and charged mutations occurred in the TM segment adjacent to the N-terminal domain (residues 24-28), while non-polar substitutions predominated in the C-terminal portion (residues 30-42). The Pro30 locus tolerated four mutations-Ala, Gly, Cys, and Ser- which represent the four side chains of least volume. Mutant coat proteins obtained directly from the phage in milligram quantities were studied by circular dichroism spectroscopy and SDS-PAGE gels. Wild type IKe coat protein solubilized in sodium deoxycholate micelles was found to occur as an alpha-helical, monomeric species which is stable at 95 degrees C, whereas the mutant Pro30-->Gly undergoes an irreversible conformational transition at ca. 90 degrees C to an aggregated beta-sheet structure. The result that Pro30 stabilizes the TM helix in the micellar membrane suggests a sterically-restricted location for the wild type Pro pyrrolidine side chain in the bulky Trp-Pro-Val triad, where it may be positioned to direct the initiation of the subsequent TM core domain helix.

  19. Structure-based design of combinatorial mutagenesis libraries.

    Science.gov (United States)

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

    2015-05-01

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

  20. Citrobacter rodentium mouse model of bacterial infection.

    Science.gov (United States)

    Crepin, Valerie F; Collins, James W; Habibzay, Maryam; Frankel, Gad

    2016-10-01

    Infection of mice with Citrobacter rodentium is a robust model to study bacterial pathogenesis, mucosal immunology, the health benefits of probiotics and the role of the microbiota during infection. C. rodentium was first isolated by Barthold from an outbreak of mouse diarrhea in Yale University in 1972 and was 'rediscovered' by Falkow and Schauer in 1993. Since then the use of the model has proliferated, and it is now the gold standard for studying virulence of the closely related human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively). Here we provide a detailed protocol for various applications of the model, including bacterial growth, site-directed mutagenesis, mouse inoculation (from cultured cells and after cohabitation), monitoring of bacterial colonization, tissue extraction and analysis, immune responses, probiotic treatment and microbiota analysis. The main protocol, from mouse infection to clearance and analysis of tissues and host responses, takes ∼5 weeks to complete.

  1. Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics.

    Science.gov (United States)

    Colson, Brett A; Thompson, Andrew R; Espinoza-Fonseca, L Michel; Thomas, David D

    2016-03-22

    We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron-electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility.

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

    Science.gov (United States)

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

    2016-05-01

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

  3. DNA MUTAGENESIS IN PANAX GINSENG CELL CULTURES

    Directory of Open Access Journals (Sweden)

    Kiselev K.V.

    2012-08-01

    Full Text Available At the present time, it is well documented that plant tissue culture induces a number of mutations and chromosome rearrangements termed “somaclonal variations”. However, little is known about the nature and the molecular mechanisms of the tissue culture-induced mutagenesis and the effects of long-term subculturing on the rate and specific features of the mutagenesis. The aim of the present study was to investigate and compare DNA mutagenesis in different genes of Panax ginseng callus cultures of different age. It has previously been shown that the nucleotide sequences of the Agrobacterium rhizogenes rolC locus and the selective marker nptII developed mutations during long-term cultivation of transgenic cell cultures of P. ginseng. In the present work, we analyzed nucleotide sequences of selected plant gene families in a 2-year-old and 20-year-old P. ginseng 1c cell culture and in leaves of cultivated P. ginseng plants. We analysed sequence variability between the Actin genes, which are a family of house-keeping genes; the phenylalanine ammonia-lyase (PAL and dammarenediol synthase (DDS genes, which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinase (SERK genes, which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-year-old callus culture was markedly higher than that in cultivated plants but lower than that in the 20-year-old callus culture of P. ginseng. Most of the mutations in the 2- and 20-year-old P. ginseng calli were A↔G and T↔C transitions. The number of nonsynonymous mutations was higher in the 2- and 20-year-old callus cultures than the number of nonsynonymous mutations in the cultivated plants of P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using methylation-sensitive DNA fragmentation

  4. Site-Directed Mutagenesis of the Virion Host Shutoff Gene (UL41) of Herpes Simplex Virus (HSV): Analysis of Functional Differences between HSV Type 1 (HSV-1) and HSV-2 Alleles

    OpenAIRE

    Everly, David N.; Read, G. Sullivan

    1999-01-01

    During lytic herpes simplex virus (HSV) infections, the HSV virion host shutoff protein (UL41) accelerates the turnover of host and viral mRNAs. Although the UL41 polypeptides from HSV type 1 (HSV-1) strain KOS and HSV-2 strain 333 are 87% identical, HSV-2 strains generally shut off the host more rapidly and completely than HSV-1 strains. In a previous study, we identified three regions of the HSV-2 UL41 polypeptide (amino acids 1 to 135, 208 to 243, and 365 to 492) that enhance the activity ...

  5. 稳定表达stathmin Ser25磷酸化位点突变的肝癌细胞株的建立%CONSTRUCTION OF STATHMIN SER25 PHOSPHORYLATION SITE-DIRECTED MUTAGENESIS HCC CELLS

    Institute of Scientific and Technical Information of China (English)

    甘淋; 陶忠桦; 刘晓燕; 刘银坤

    2011-01-01

    目的:建立稳定表达stathmin Ser25磷酸化位点点突变(stathmin S25A)的肝癌细胞株.方法:采用PCR定点突变的方法构建stathmin S25A的重组质粒,并用酶切和测序技术鉴定突变结果;采用脂质体转染的方法,筛选建立stathmin野生型(wt)和突变型(S25A)的肝癌细胞HCCLM6,用Western blotting进行鉴定.结果:定点突变构建stathmin S25A的重组质粒,测序证实stathmin 25位丝氨酸突变为丙氨酸.将stathmin wt和S25A转染HCCLM6,筛选建立稳定表达的肝癌细胞株,Western blotting检测发现stathmin pS25在stathmin S25A细胞中的表达较stathmin wt和对照组细胞明显下降(P<0.05).结论:建立了稳定表达stathmin S25A的肝癌细胞株,为研究stathmin位点磷酸化在肝癌发病中的分子机制提供实验模型.

  6. Calcineurin A versus NS5A-TP2/HD domain containing 2: a case study of site-directed low-frequency random mutagenesis for dissecting target specificity of peptide aptamers.

    Science.gov (United States)

    Dibenedetto, Silvia; Cluet, David; Stebe, Pierre-Nicolas; Baumle, Véronique; Léault, Jérémie; Terreux, Raphaël; Bickle, Marc; Chassey, Benoit D E; Mikaelian, Ivan; Colas, Pierre; Spichty, Martin; Zoli, Michele; Rudkin, Brian B

    2013-07-01

    We previously identified a peptide aptamer (named R5G42) via functional selection for its capacity to slow cell proliferation. A yeast two-hybrid screen of human cDNA libraries, using R5G42 as "bait," allowed the identification of two binding proteins with very different functions: calcineurin A (CnA) (PP2B/PPP3CA), a protein phosphatase well characterized for its role in the immune response, and NS5A-TP2/HD domain containing 2, a much less studied protein induced subsequent to hepatitis C virus non-structural protein 5A expression in HepG2 hepatocellular carcinoma cells, with no known activity. Our objective in the present study was to dissect the dual target specificity of R5G42 in order to have tools with which to better characterize the actions of the peptide aptamers toward their individual targets. This was achieved through the selection of random mutants of the variable loop, derived from R5G42, evaluating their specificity toward CnA and NS5A-TP2 and analyzing their sequence. An interdisciplinary approach involving biomolecular computer simulations with integration of the sequence data and yeast two-hybrid binding phenotypes of these mutants yielded two structurally distinct conformers affording the potential molecular basis of the binding diversity of R5G42. Evaluation of the biological impact of CnA- versus NS5A-TP2-specific peptide aptamers indicated that although both contributed to the anti-proliferative effect of R5G42, CnA-binding was essential to stimulate the nuclear translocation of nuclear factor of activated T cells, indicative of the activation of endogenous CnA. By dissecting the target specificity of R5G42, we have generated novel tools with which to study each target individually. Apta-C8 is capable of directly activating CnA independent of binding to NS5A-TP2 and will be an important tool in studying the role of CnA activation in the regulation of different signaling pathways, whereas Apta-E1 will allow dissection of the function of NS5A-TP2, serving as an example of the usefulness of peptide aptamer technology for investigating signaling pathways.

  7. Structural and mechanistic analysis of trichodiene synthase using site-directed mutagenesis: probing the catalytic function of tyrosine-295 and the asparagine-225/serine-229/glutamate-233-Mg2+B motif.

    Science.gov (United States)

    Vedula, L Sangeetha; Jiang, Jiaoyang; Zakharian, Tatiana; Cane, David E; Christianson, David W

    2008-01-15

    Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the "aspartate-rich" motif D(100)DXX(D/E) that coordinates to Mg2+A and Mg2+C, and the "NSE/DTE" motif N(225)DXXSXXXE that chelates Mg2+B (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like DDXXTXXXE motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpene products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.

  8. Site-directed mutagenesis of HIV-1 vpu gene demonstrates two clusters of replication-defective mutants with distinct ability to down-modulate cell surface CD4 and tetherin

    Directory of Open Access Journals (Sweden)

    Masako Nomaguchi

    2010-11-01

    Full Text Available HIV-1 Vpu acts positively on viral infectivity by mediating CD4 degradation in endoplasmic reticulum and enhances virion release by counteracting a virion release restriction factor, tetherin. In order to define the impact of Vpu activity on HIV-1 replication, we have generated a series of site-specific proviral vpu mutants. Of fifteen mutants examined, seven exhibited a replication-defect similar to that of a vpu-deletion mutant in a lymphocyte cell line H9. These mutations clustered in narrow regions within transmembrane domain (TMD and cytoplasmic domain (CTD. Replication-defective mutants displayed the reduced ability to enhance virion release from a monolayer cell line HEp2 without exception. Upon transfection with Vpu expression vectors, neither TMD mutants nor CTD mutants blocked CD4 expression at the cell surface in another monolayer cell line MAGI. While TMD mutants were unable to down-modulate cell surface tetherin in HEp2 cells, CTD mutants did quite efficiently. Confocal microscopy analysis revealed the difference of intracellular localization between TMD and CTD mutants. In total, replication capability of HIV-1 carrying vpu mutations correlates well with the ability of Vpu to enhance virion release and to impede the cell surface expression of CD4 but not with the ability to down-modulate cell surface tetherin. Our results here suggest that efficient viral replication requires not only down-regulation of cell surface tetherin but also its degradation.

  9. Site Directed Mutagenesis for C/EBPα Binding Sites in Chicken L-FABP Promoter%鸡L-FABP启动子区C/EBPα结合位点的定点突变分析

    Institute of Scientific and Technical Information of China (English)

    贺綦; 孙婴宁; 李辉; 王启贵

    2015-01-01

    为进一步分析L-FABP启动子区域中的C/EBPα结合位点以确定其在L-FABP转录中的调控作用.本研究采用定点突变方法将L-FABP启动子区域中的C/EBPα结合位点进行有效突变.结果显示,C/EBPα外源过表达可以抑制L-FABP启动子活性,突变L-FABP启动子区域中C/EBPα结合位点后L-FABP的启动子活性明显升高.这些结果表明,鸡L-FABP基因受到C/EBPα基因负调控作用,且C/EBPα很可能通过与该位点结合参与L-FABP的转录调控,为进一步研究C/EBPα在L FABP基因转录调控中的作用提供重要依据.

  10. 定点突变提高苯丙氨酸羟化酶的热稳定性%Thermal stability improvement for phenylalanine hydroxylase by site-directed mutagenesis

    Institute of Scientific and Technical Information of China (English)

    叶双双; 周丽; 周哲敏

    2016-01-01

    嗜热菌中,蛋白质存在Ala替换Gly以及Arg替换Lys的趋势.为了提高紫色色杆菌来源的苯丙氨酸羟化酶的热稳定性,将该酶中所有Gly突变成Ala,tys突变成Arg,筛选获得热稳定性提高的突变体,并进行组合突变,对突变酶的酶学性质进行研究.结果表明,突变酶K94R和G221A在50℃的半衰期分别为26.2 min、16.8 min,比原始酶(9.0 min)分别提高了1.9倍、0.9倍,同时组合突变酶K94R/G221A在50℃处理1h后仍保留65.6%的酶活,比原始酶(8.6%)高出6.6倍.圆二色谱结果显示原始酶和突变酶K94R、G221A及K94R/G221A的Tm值分别为51.5℃、53.8℃、53.1℃和54.8℃.蛋白三维结构模拟推测突变体热稳定性提高机理为:突变体K94R中Arg94与Ile95之间形成额外氢键,稳定其所在的柔性区域;突变体G221A中Ala221与Leu281产生疏水作用,稳定酶分子C-端柔性区.该研究结果为蛋白质热稳定性改造提供了参考,也为苯丙氨酸羟化酶在功能性食品领域的应用奠定了基础.

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

    Directory of Open Access Journals (Sweden)

    Guyue Cheng

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

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

    Science.gov (United States)

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

    2016-02-01

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

  13. Cell-mediated mutagenesis by chemical carcinogens

    Energy Technology Data Exchange (ETDEWEB)

    Huberman, E.; Langenbach, R.

    1978-01-01

    The cell-mediated mutation system, with the proper choice of metabolizing cells, can be used to detect the mutagenic activities of different classes of chemical carcinogens. When fibroblastic cells were used as the metabolizing cells, a correlation between the in vivo carcinogenic activity and the in vitro mutagenic activity of 11 aromatic polycyclic hydrocarbons was observed. When primary liver cells were used as the metabolizing cells, three known liver carcinogens were demonstrated to be mutagenic by the cell-mediated assay, while two non-carcinogenic analogues were not mutagenic. These results from the cell-mediated system suggest that the reactive intermediates of the carcinogens are stable enough to be transferred from the metabolizing cells to the V79 cells. The cell-mediated mutagenesis system is a simple in vitro assay which may simulate the in vivo situation. It was concluded that this approach could be extended to the co-cultivation of cells from other organs or tissues with mutable mammalian cells.

  14. Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals.

    NARCIS (Netherlands)

    van Boxtel, R.; Toonen, P.W.; Verheul, M.; van Roekel, H.S.; Nijman, I.J.; Guryev, V.; Cuppen, E.

    2008-01-01

    BACKGROUND: The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is cur

  15. Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals

    NARCIS (Netherlands)

    van Boxtel, Ruben; Toonen, Pim W; Verheul, Mark; van Roekel, Henk S; Nijman, Isaac J; Guryev, Victor; Cuppen, Edwin

    2008-01-01

    BACKGROUND: The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is cur

  16. Mutagenesis of Ser24 of cytochrome b559 α subunit affects PSⅡ activities in Chlamydomonas reinhardtii

    Institute of Scientific and Technical Information of China (English)

    MA JingJing; LI LiangBi; JING YuXiang; KUANG TingYun

    2007-01-01

    In order to study the functions of cytochrome b559 (Cyt b559) in photosystem two (PSⅡ) activity, mutant S24F of Chlamydomonas reinhardtii was constructed using site directed mutagenesis, in which Serine24 (Ser24) locating downstream of Histidine23 (His23) in c subunit of Cyt b559 was replaced by Phenylalanine (Phe). Physiological and biochemical analysis showed that mutant S24F could be grown photoautotrophically or photoheterotrophically. However, their growth rate was slower either on HSM or TAP medium than that of the control; Analysis of PSⅡ activity revealed that its oxygen evolution was about 71% of wild type (WT); The Photochemical efficiency of PSll (Fv/Fm) of S24F was reduced 0.23 compared with WT; S24F was more sensitive to strong light irradiance than the wild type; Furthermore,SDS-PAGE and Western-blotting analysis indicated that the expression levels of c subunit of Cyt b559,LHCⅡ and PsbO of S24F were a little less than those of the wild type. Overall, these data suggests that Ser24 plays a significant role in making Cyt b559 structure maintain PSⅡ complex activity of oxygen evolution although it is not directly bound to heme group.

  17. Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening.

    Science.gov (United States)

    Molinski, Steven V; Ahmadi, Saumel; Hung, Maurita; Bear, Christine E

    2015-12-01

    There are nearly 2000 mutations in the CFTR gene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mutations in the CFTR gene. This method was used to generate CFTR mutants bearing deletions (p.Gln2_Trp846del, p.Ser700_Asp835del, p.Ile1234_Arg1239del) and truncation with polyhistidine tag insertion (p.Glu1172-3Gly-6-His*), which either recapitulate a disease phenotype or render tools for modulator binding site identification, with subsequent evaluation of drug responses using a high-throughput (384-well) membrane potential-sensitive fluorescence assay of CFTR channel activity within a 1 wk time frame. This proof-of-concept study shows that these methods enable rapid and quantitative comparison of multiple CFTR mutants to emerging drugs, facilitating future large-scale efforts to stratify mutants according to their "theratype" or most promising targeted therapy.

  18. 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.

  19. 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.

  20. Rational Mutagenesis of Cyclodextrin Glucanotransferase at the Calcium Binding Regions for Enhancement of Thermostability

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    Kian Mau Goh

    2012-04-01

    Full Text Available Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as “parent CGTase” herein. Four CGTase variants (S182G, S182E, N132R and N28R were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.

  1. Rational mutagenesis of cyclodextrin glucanotransferase at the calcium binding regions for enhancement of thermostability.

    Science.gov (United States)

    Goh, Poh Hong; Illias, Rosli Md; Goh, Kian Mau

    2012-01-01

    Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as "parent CGTase" herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.

  2. 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.

  3. 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.

  4. 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)

  5. Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin

    Science.gov (United States)

    Toniti, Waraphan; Yoshida, Toru; Tsurumura, Toshiharu; Irikura, Daisuke; Monma, Chie; Kamata, Yoichi

    2017-01-01

    Unusual outbreaks of food poisoning in Japan were reported in which Clostridium perfringens was strongly suspected to be the cause based on epidemiological information and fingerprinting of isolates. The isolated strains lack the typical C. perfringens enterotoxin (CPE) but secrete a new enterotoxin consisting of two components: C. perfringens iota-like enterotoxin-a (CPILE-a), which acts as an enzymatic ADP-ribosyltransferase, and CPILE-b, a membrane binding component. Here we present the crystal structures of apo-CPILE-a, NAD+-CPILE-a and NADH-CPILE-a. Though CPILE-a structure has high similarity with known iota toxin-a (Ia) with NAD+, it possesses two extra-long protruding loops from G262-S269 and E402-K408 that are distinct from Ia. Based on the Ia–actin complex structure, we focused on actin-binding interface regions (I-V) including two protruding loops (PT) and examined how mutations in these regions affect the ADP-ribosylation activity of CPILE-a. Though some site-directed mutagenesis studies have already been conducted on the actin binding site of Ia, in the present study, mutagenesis studies were conducted against both α- and β/γ-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both α- and β/γ-actin, but its sensitivity towards β/γ-actin is 36% compared with α-actin. Our results contrast to that only C2-I ADP-ribosylates β/γ-actin. We also showed that PT-I and two convex-concave interactions in CPILE-a are important for actin binding. The current study is the first detailed analysis of site-directed mutagenesis in the actin binding region of Ia and CPILE-a against both α- and β/γ-actin. PMID:28199340

  6. Efficient Targeted Mutagenesis in Medaka Using Custom-Designed Transcription Activator-Like Effector Nucleases

    OpenAIRE

    Ansai, Satoshi; Sakuma, Tetsushi; Yamamoto, Takashi; Ariga, Hiroyoshi; Uemura, Norihito; Takahashi, Ryosuke; Kinoshita, Masato

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) have become powerful tools for targeted genome editing. Here we demonstrate efficient targeted mutagenesis in medaka (Oryzias latipes), which serves as an excellent vertebrate model for genetics and genomics. We designed and constructed a pair of TALENs targeting the medaka DJ-1 gene, a homolog of human DJ-1 (PARK7). These TALENs induced a number of insertions and deletions in the injected embryos with extremely high efficiency. This in...

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    E. Wyroba

    2016-04-01

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

  9. Endogenous mutagenesis in recombinant sulfolobus plasmids.

    Science.gov (United States)

    Sakofsky, Cynthia J; Grogan, Dennis W

    2013-06-01

    Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the β-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac(-) mutants also grew faster than the Lac(+) parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 × 10(-4) mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G · C-to-A · T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.

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

    Science.gov (United States)

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

    2001-04-01

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

  11. Development of Designed Site-Directed Pseudopeptide-Peptido-Mimetic Immunogens as Novel Minimal Subunit-Vaccine Candidates for Malaria

    Directory of Open Access Journals (Sweden)

    Luisa F. Carreño

    2010-12-01

    Full Text Available Synthetic vaccines constitute the most promising tools for controlling and preventing infectious diseases. When synthetic immunogens are designed from the pathogen native sequences, these are normally poorly immunogenic and do not induce protection, as demonstrated in our research. After attempting many synthetic strategies for improving the immunogenicity properties of these sequences, the approach consisting of identifying high binding motifs present in those, and then performing specific changes on amino-acids belonging to such motifs, has proven to be a workable strategy. In addition, other strategies consisting of chemically introducing non-natural constraints to the backbone topology of the molecule and modifying the α-carbon asymmetry are becoming valuable tools to be considered in this pursuit. Non-natural structural constraints to the peptide backbone can be achieved by introducing peptide bond isosters such as reduced amides, partially retro or retro-inverso modifications or even including urea motifs. The second can be obtained by strategically replacing L-amino-acids with their enantiomeric forms for obtaining both structurally site-directed designed immunogens as potential vaccine candidates and their Ig structural molecular images, both having immuno-therapeutic effects for preventing and controlling malaria.

  12. Site directed spin labelling and pulsed dipolar electron paramagnetic resonance (double electron-electron resonance) of force activation in muscle

    Energy Technology Data Exchange (ETDEWEB)

    Fajer, Piotr G [Institute of Molecular Biophysics, Department of Biological Science, National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310 (United States)

    2005-05-11

    The recent development of site specific spin labelling and advances in pulsed electron paramagnetic resonance(EPR) have established spin labelling as a viable structural biology technique. Specific protein sites or whole domains can be selectively targeted for spin labelling by cysteine mutagenesis. The secondary structure of the proteins is determined from the trends in EPR signals of labels attached to consecutive residues. Solvent accessibility or label mobility display periodicities along the labelled polypeptide chain that are characteristic of {beta}-strands (periodicity of 2 residues) or {alpha}-helices (3.6 residues). Low-resolution 3D structure of proteins is determined from the distance restraints. Two spin labels placed within 60-70 A of each other create a local dipolar field experienced by the other spin labels. The strength of this field is related to the interspin distance, {proportional_to} r{sup -3}. The dipolar field can be measured by the broadening of the EPR lines for the short distances (8-20 A) or for the longer distances (17-70 A) by the pulsed EPR methods, double electron-electron resonance(DEER) and double quantum coherence (DQC). A brief review of the methodology and its applications to the multisubunit muscle protein troponin is presented below.

  13. 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.

  14. 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.

  15. 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.

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

    Science.gov (United States)

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

    2014-01-01

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

  17. Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells.

    Science.gov (United States)

    Ivics, Zoltán; Izsvák, Zsuzsanna; Medrano, Gerardo; Chapman, Karen M; Hamra, F Kent

    2011-09-08

    We describe an experimental approach for generating mutant alleles in rat spermatogonial stem cells (SSCs) using Sleeping Beauty (SB) transposon-mediated insertional mutagenesis. The protocol is based on mobilization of mutagenic gene-trap transposons from transfected plasmid vectors into the genomes of cultured stem cells. Cells with transposon insertions in expressed genes are selected on the basis of activation of an antibiotic-resistance gene encoded by the transposon. These gene-trap clones are transplanted into the testes of recipient males (either as monoclonal or polyclonal libraries); crossing of these founders with wild-type females allows the insertions to be passed to F(1) progeny. This simple, economic and user-friendly methodological pipeline enables screens for functional gene annotation in the rat, with applicability in other vertebrate models where germ line-competent stem cells have been established. The complete protocol from transfection of SSCs to the genotyping of heterozygous F(1) offspring that harbor genomic SB gene-trap insertions takes 5-6 months.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. 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.

  20. Functional studies of the gene slr2049 from Synechocystis sp. PCC6803 and its site-directed mutation.

    Science.gov (United States)

    Liu, Bingjun; Chen, Sili; Zhang, Lei

    2015-06-01

    Phycobiliprotein is a homologous family of light-harvesting chromoproteins existing in cyanobacteria, red algae and cryptophytes. Phycobiliprotein is made up of phycobilin and its corresponding apophycobiliprotein, and they are covalently linked by the thioether bond with the bilin lyase. Using the software BLAST, we have found gene slr2049 in Synechocystis sp. PCC6803 homologous to the biliprotein lyase gene cpeS. This paper investigates the protein expressed by gene slr2049 to find the enzymatic activity characteristics. We cloned slr2049 and its related genes cpcB, ho1, and pcyA which are linked with the synthesis of phycocyanin. Special amino acid mutagenesis was performed on slr2049 to construct eight mutants slr2049 (H21S), slr2049 (L23S), slr2049 (A24S), slr2049 (F25S), slr2049 (W72L), slr2049 (G84S), slr2049 (R107S) and slr2049 (Y124S). These mutants were ligated with vectors pEDFDuet-1 and pET-23a to construct pCDF-cpcB-slr2049 wild-type, pCDF-cpcB-slr2049 mutants and pET-ho1-pcyA, for the purpose of protein expression and analysis. The results showed that the wild-type and mutants slr2049 (H21S), slr2049 (L23S), slr2049 (F25S), slr2049 (W72L), slr2049 (G84S), and slr2049 (Y124S) can catalyze CpcB to couple on PCB correctly and the products have unique spectral characteristics. However mutants slr2049 (A24S) and slr2049 (R107S) have no spectral characteristics. Thus, it is suggested that alanine at position 24 and arginine at position 107 are the active sites.

  1. 2012 MUTAGENESIS GORDON RESEARCH CONFERENCE, AUGUST 19-23, 2012

    Energy Technology Data Exchange (ETDEWEB)

    Demple, Bruce

    2012-08-23

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

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

    Science.gov (United States)

    Kozmin, Stanislav G; Jinks-Robertson, Sue

    2013-03-01

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

  3. 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.

  4. Global structure of a three-way junction in a phi29 packaging RNA dimer determined using site-directed spin labeling.

    Science.gov (United States)

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

    2012-02-08

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-08

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

  6. Genetic aspects of targeted insertion mutagenesis in yeasts.

    Science.gov (United States)

    Klinner, U; Schäfer, B

    2004-05-01

    Targeted insertion mutagenesis is a main molecular tool of yeast science initially applied in Saccharomyces cerevisiae. The method was extended to fission yeast Schizosaccharomyces pombe and to "non-conventional" yeast species, which show specific properties of special interest to both basic and applied research. Consequently, the behaviour of such non-Saccharomyces yeasts is reviewed against the background of the knowledge of targeted insertion mutagenesis in S. cerevisiae. Data of homologous integration efficiencies obtained with circular, ends-in or ends-out vectors in several yeasts are compared. We follow details of targeted insertion mutagenesis in order to recognize possible rate-limiting steps. The route of the vector to the target and possible mechanisms of its integration into chromosomal genes are considered. Specific features of some yeast species are discussed. In addition, similar approaches based on homologous recombination that have been established for the mitochondrial genome of S. cerevisiae are described.

  7. Hapten-directed spontaneous disulfide shuffling: a universal technology for site-directed covalent coupling of payloads to antibodies

    Science.gov (United States)

    Dengl, Stefan; Hoffmann, Eike; Grote, Michael; Wagner, Cornelia; Mundigl, Olaf; Georges, Guy; Thorey, Irmgard; Stubenrauch, Kay-Gunnar; Bujotzek, Alexander; Josel, Hans-Peter; Dziadek, Sebastian; Benz, Joerg; Brinkmann, Ulrich

    2015-01-01

    ., Georges, G., Thorey, I., Stubenrauch, K.-G., Bujotzek, A., Josel, H.-P., Dziadek, S., Benz, J., Brinkmann, U. Hapten-directed spontaneous disulfide shuffling: a universal technology for site-directed covalent coupling of payloads to antibodies. PMID:25670234

  8. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling

    Science.gov (United States)

    Evans, Eric G.B.; Millhauser, Glenn L.

    2016-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain “K1,” and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron–electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite

  9. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling.

    Science.gov (United States)

    Evans, Eric G B; Millhauser, Glenn L

    2015-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain "K1," and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron-electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite reagents

  10. Mutagenesis of Trichoderma Viride by Ultraviolet and Plasma

    Science.gov (United States)

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

    2012-04-01

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

  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 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.

  12. Mutagenesis of Trichoderma Viride by Ultraviolet and Plasma

    Institute of Scientific and Technical Information of China (English)

    姚日生; 李曼曼; 邓胜松; 胡华佳; 王淮; 李凤和

    2012-01-01

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

  13. 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.

  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. 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

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

    Science.gov (United States)

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

    2015-05-07

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

  17. 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.

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

    Science.gov (United States)

    Linde, Ana R.; Garcia-Vazquez, Eva

    2009-01-01

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

  19. 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...

  20. A simple mutagenesis using natural competence in Tannerella forsythia.

    Science.gov (United States)

    Nishikawa, Kiyoshi; Tanaka, Yoshinobu

    2013-09-01

    We report the discovery of natural competence in Tannerella forsythia and its application to targeted chromosomal mutagenesis. Keeping T. forsythia in a biofilm throughout the procedure allowed efficient DNA uptake and allelic replacement. This simple method is cost-effective and reproducible compared with the conventional protocols using broth culture and electroporation.

  1. 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.

  2. Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Kai, Mihoko; Wang, Teresa S.-F

    2003-11-27

    Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Pol{kappa}). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks. Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development.

  3. RNA-Guided Cas9-Induced Mutagenesis in Tobacco Followed by Efficient Genetic Fixation in Doubled Haploid Plants

    Science.gov (United States)

    Schedel, Sindy; Pencs, Stefanie; Hensel, Götz; Müller, Andrea; Rutten, Twan; Kumlehn, Jochen

    2017-01-01

    Customizable endonucleases are providing an effective tool for genome engineering. The resulting primary transgenic individuals (T0) are typically heterozygous and/or chimeric with respect to any mutations induced. To generate genetically fixed mutants, they are conventionally allowed to self-pollinate, a procedure which segregates individuals into mutant heterozygotes/homozygotes and wild types. The chances of recovering homozygous mutants among the progeny depend not only on meiotic segregation but also on the frequency of mutated germline cells in the chimeric mother plant. In Nicotiana species, the heritability of Cas9-induced mutations has not been demonstrated yet. RNA-guided Cas9 endonuclease-mediated mutagenesis was targeted to the green fluorescent protein (GFP) gene harbored by a transgenic tobacco line. Upon retransformation using a GFP-specific guide RNA/Cas9 construct, the T0 plants were allowed to either self-pollinate, or were propagated via regeneration from in vitro cultured embryogenic pollen which give rise to haploid/doubled haploid plants or from leaf explants that form plants vegetatively. Single or multiple mutations were detected in 80% of the T0 plants. About half of these mutations proved heritable via selfing. Regeneration from in vitro cultured embryogenic pollen allowed for homozygous mutants to be produced more efficiently than via sexual reproduction. Consequently, embryogenic pollen culture provides a convenient method to rapidly generate a variety of genetically fixed mutants following site-directed mutagenesis. The recovery of a mutation not found among sexually produced and analyzed progeny was shown to be achievable through vegetative plant propagation in vitro, which eventually resulted in heritability when the somatic clones were selfed. In addition, some in-frame mutations were associated with functional attenuation of the target gene rather than its full knock-out. The generation of mutants with compromised rather than

  4. 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.

  5. 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 m

  6. Structure-Function Relationship of a Plant NCS1 Member - Homology Modeling and Mutagenesis Identified Residues Critical for Substrate Specificity of PLUTO, a Nucleobase Transporter from Arabidopsis: e91343

    National Research Council Canada - National Science Library

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

    2014-01-01

    .... We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens...

  7. Generation of mutants with developmental defects in zebrafish by ENU mutagenesis

    Institute of Scientific and Technical Information of China (English)

    JIN Peng; TIAN Tian; SUN Zhihui; MENG Anming

    2004-01-01

    As a good model for studying early development of vertebrates, zebrafish (Danio rerio) is attracting more and more attention. Following ENU mutagenesis, 320 F2 families were established. Mutants, which showed defects in epiboly, axis, somite, head, and cardiac and blood systems, were identified by observing morphological changes in F3 embryos. So far, 35 mutant lines have been established, the majority of which showed anomalies in axis and somite formation. These mutant lines provide useful genetic resources for cloning of the mutant genes and for studying mechanisms of early development of vertebrate embryos.

  8. Sulfyhydryl-reactive site-directed cross-linking as a method for probing the tetrameric structure of histones H3 and H4.

    Science.gov (United States)

    Bowman, Andrew; Owen-Hughes, Tom

    2012-01-01

    The structural characterisation of protein-protein interactions is often challenging. Where interactions are not amenable to high-resolution approaches, alternatives providing lower resolution information are often of value. One such approach is site-directed cross-linking. Here, through the introduction of cysteine residues at strategic locations in histone proteins, we use site-directed cross-linking to monitor the association of chromatin subunits. This approach is informative for the study of both recombinant and native chromatin complexes consisting either of histone subunits alone or in association with accessory proteins, in this case histone chaperones. The approaches described may be generally applicable for monitoring the interactions of a diverse range of multi-protein complexes.

  9. Mutation induced extinction in finite populations: lethal mutagenesis and lethal isolation.

    Directory of Open Access Journals (Sweden)

    C Scott Wylie

    Full Text Available Reproduction is inherently risky, in part because genomic replication can introduce new mutations that are usually deleterious toward fitness. This risk is especially severe for organisms whose genomes replicate "semi-conservatively," e.g. viruses and bacteria, where no master copy of the genome is preserved. Lethal mutagenesis refers to extinction of populations due to an unbearably high mutation rate (U, and is important both theoretically and clinically, where drugs can extinguish pathogens by increasing their mutation rate. Previous theoretical models of lethal mutagenesis assume infinite population size (N. However, in addition to high U, small N can accelerate extinction by strengthening genetic drift and relaxing selection. Here, we examine how the time until extinction depends jointly on N and U. We first analytically compute the mean time until extinction (τ in a simplistic model where all mutations are either lethal or neutral. The solution motivates the definition of two distinct regimes: a survival phase and an extinction phase, which differ dramatically in both how τ scales with N and in the coefficient of variation in time until extinction. Next, we perform stochastic population-genetics simulations on a realistic fitness landscape that both (i features an epistatic distribution of fitness effects that agrees with experimental data on viruses and (ii is based on the biophysics of protein folding. More specifically, we assume that mutations inflict fitness penalties proportional to the extent that they unfold proteins. We find that decreasing N can cause phase transition-like behavior from survival to extinction, which motivates the concept of "lethal isolation." Furthermore, we find that lethal mutagenesis and lethal isolation interact synergistically, which may have clinical implications for treating infections. Broadly, we conclude that stably folded proteins are only possible in ecological settings that support sufficiently

  10. Multi-fragment site-directed mutagenic overlap extension polymerase chain reaction as a competitive alternative to the enzymatic assembly method.

    Science.gov (United States)

    Wäneskog, Marcus; Bjerling, Pernilla

    2014-01-01

    Methods for introducing multiple site-directed mutations are important experimental tools in molecular biology. Research areas that use these methods include the investigation of various protein modifications in cellular processes, modifying proteins for efficient recombinant expression, and the stabilization of mRNAs to allow for increased protein expression. Introducing multiple site-directed mutations is also an important tool in the field of synthetic biology. There are two main methods used in the assembling of fragments generated by mutagenic primers: enzymatic assembly and overlap extension polymerase chain reaction (OE-PCR). In this article, we present an improved OE-PCR method that can be used for the generation of large DNA fragments (up to 7.4 kb) where at least 13 changes can be introduced using a genomic template. The improved method is faster (due to fewer reaction steps) and more accurate (due to fewer PCR cycles), meaning that it can effectively compete with the enzymatic assembly method. Data presented here show that the site-directed mutations can be introduced anywhere between 50 and 1800 bp from each other. The method is highly reliable and predicted to be applicable to most DNA engineering when the introduction of multiple changes in a DNA sequence is required.

  11. 利用 TALEN 技术建立恒河猴 TRIM5α基因 突变细胞株%Establishment of Mutagenesis Cell Line of Macaca mulatta Gene TRIM5α by TALEN

    Institute of Scientific and Technical Information of China (English)

    王小莉; 余庆; 袁雅红; 腾智平; 李东升; 曾毅

    2016-01-01

    利用 TALEN 技术建立恒河猴 TRIM5α基因突变细胞株,为进一步研究 TRIM5α基因的功能奠定基础。构建针对TRIM5α打靶基因的 TALEN,通过点突变的方法获得包含 TRIM5α第7个外显子中打靶位点1211-1224的基因序列并构建点突变donor 载体。将打靶 TRIM5α基因的 TALEN 质粒和 donor 质粒电转入恒河猴肾细胞(LLC-MK2)中,无限稀释法获得单克隆细胞系,通过抽提基因组 DNA,再利用 PCR 技术扩增目标序列并测序筛选出 TRIM5α碱基缺失(1215-1216)和点突变(1213-1215,1217)的细胞系。通过共转 TALEN 质粒和点突变的 donor 质粒筛选获得了 TRIM5α基因敲除和 TRIM5α基因点突变的 LLC-MK2细胞系。%Using transcription activator-like effector nuclease(TALEN)to establish the mutagenesis cell line of Macaca mulatta gene TRIM5α is to lay the foundation for further studying the function of TRIM5α gene. The TALEN plasmids targeting TRIM5α was constructed. The site-directed gene mutagenesis donor vector was obtained by site-directed gene mutagenesis technology and the sequence contained the seventh exon of TRIM5α at the position from 1 211 to 1 224. The TALEN plasmids and donor vector were co-transfected into the M. mulatta’s kidney cell line(LLC-MK2)by electroporation. The single cell line was obtained by the infinite dilution method. By extracting the genomic DNA and amplifying the target sequence by PCR,the cell line with base deletion(1 215-1 216)and site mutations(1 213-1 215,1 217)of TRIM5α was screened. Conclusively,the LLC-MK2 single cell line with knockout and site-directed gene mutagenesis of gene TRIM5α was obtained.

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

    Science.gov (United States)

    Sun, Dawei; Ostermaier, Martin K; Heydenreich, Franziska M; Mayer, Daniel; Jaussi, Rolf; Standfuss, Joerg; Veprintsev, Dmitry B

    2013-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Weina; Hellinga, Homme W.; Beese, Lorena S. (Duke)

    2012-05-10

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

  14. Selection acts on DNA secondary structures to decrease transcriptional mutagenesis.

    Directory of Open Access Journals (Sweden)

    Claire Hoede

    2006-11-01

    Full Text Available Single-stranded DNA is more subject to mutation than double stranded. During transcription, DNA is transiently single stranded and therefore subject to higher mutagenesis. However, if local intra-strand secondary structures are formed, some bases will be paired and therefore less sensitive to mutation than unpaired bases. Using complete genome sequences of Escherichia coli, we show that local intra-strand secondary structures can, as a consequence, be used to define an index of transcription-driven mutability. At gene level, we show that natural selection has favoured a reduced transcription-driven mutagenesis via the higher than expected frequency of occurrence of intra-strand secondary structures. Such selection is stronger in highly expressed genes and suggests a sequence-dependent way to control mutation rates and a novel form of selection affecting the evolution of synonymous mutations.

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

    Directory of Open Access Journals (Sweden)

    Kentaro Ishida

    2015-10-01

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

  16. Insertional mutagenesis of an industrial strain of Streptococcus thermophilus.

    Science.gov (United States)

    Labarre, C; Schirawski, J; van der Zwet, A; Fitzgerald, G F; van Sinderen, D

    2001-06-12

    Random mutagenesis of an industrial strain of Streptococcus thermophilus was achieved through an adapted version of a two-plasmid system. The mutagenesis strategy is based on random integration of derivatives of the non-replicative (Rep(-)) plasmid pORI19 by means of homologous recombination following a temperature shift that eliminates replication of the temperature-sensitive (Rep(ts)) helper plasmid pVE6007. In this way mutants were generated which were affected in bacteriophage sensitivity or sucrose metabolism. Homologues were identified of a protein related to folate metabolism from a bacteriophage-resistant mutant and of two subunits of an oligopeptide transport system from a mutant deficient in sucrose utilisation.

  17. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-01-27

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

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

    Science.gov (United States)

    Schwartz, Jeffrey L

    2017-07-25

    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. Published by Elsevier B.V.

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

    Directory of Open Access Journals (Sweden)

    Dominique H Limoli

    2014-04-01

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

  1. Virtual mutagenesis of isocitrate dehydrogenase 1 involved in glioblastoma multiforme

    Institute of Scientific and Technical Information of China (English)

    WANG Ming-dong; SHI Yan-fang; WANG Hong; WANG Jia-liang; MA Wen-bin; WANG Ren-zhi

    2011-01-01

    Background Site A132Arg mutations potentially impair the affinity of isocitrate dehydrogenase 1 (IDH1) for its substrate isocitrate (ICT),consequently reducing the production of α-ketoglutarate and leading to tumor growth through the induction of the hypoxia-inducible factor-1 (HIF-1) pathway.However,given that the roles of other active sites in IDH1 substrate binding remain unclear,we aimed to investigate IDH1 mutation pattern and its influence on enzyme function.Methods Fifteen IDH1 catalytic active site candidates were selected for in silico mutagenesis and protein homology modeling.Binding free energy of the IDH1/ICT complexes with single-site mutations was compared with that of the wild type.The affinity of 10 IDH1 catalytic active sites for the ICT substrate was further calculated.Results The IDH1 active site included seven residues from chain A (A77Thr,A94Ser,A100Arg,A132Arg,A1O9Arg,A275Asp,and A279Asp) and three residues from chain B (B214Thr,B212Lys,and B252Asp) that constituted the substrate ICT-binding site.These residues were located within 0.5 nm of ICT,indicating a potential interaction with the substrate.IDH1 changes of binding free energy (△E) suggested that the A132Arg residue from chain A contributes three hydrogen bonds to the ICT α-carboxyl and β-carboxyl groups,while the other nine residues involved in ICT binding form only one or two hydrogen bonds.Amino acid substitutes at A132Arg,A109Arg,and B212Lys sites,had the greatest effect on enzyme affinity for its substrate.Conclusions Mutations at sites A132Arg,A109Arg,and B212Lys reduced IDH1 affinity for ICT,indicating these active sites may play a central role in substrate binding.Mutations at sites A77Thr,A94Ser,and A275Asp increased the affinity of IDH1 for ICT,which may enhance IDN1 catalytic activity.Mutant IDH1 proteins with higher catalytic activity than the wild-type IDH1 could potentially be used as a novel gene therapy for glioblastoma multiforme.

  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.

  3. Molecular characterization of chemical mutagenesis induced diversity in elite maize germplasm

    Directory of Open Access Journals (Sweden)

    Christov Nikolai K.

    2004-01-01

    -105, as well as in the classical breeding line B73. The data, presented here demonstrate the usefulness of chemical mutagenesis for generation of genetic diversity within the elite maize germplasm. Some of this variation may affect the major genes in the QTLs. Our initial data revealed mutagenesis induced polymorphisms in the coding sequences of two important for the determination of flowering time transcription factors. Further molecular analyses of the proposed model systems may complement the trait association efforts and will help to directly identify the major genes in the QTLs.

  4. 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

  5. Characterisation of a C1qtnf5 Ser163Arg knock-in mouse model of late-onset retinal macular degeneration.

    Directory of Open Access Journals (Sweden)

    Xinhua Shu

    Full Text Available A single founder mutation resulting in a Ser163Arg substitution in the C1QTNF5 gene product causes autosomal dominant late-onset retinal macular degeneration (L-ORMD in humans, which has clinical and pathological features resembling age-related macular degeneration. We generated and characterised a mouse "knock-in" model carrying the Ser163Arg mutation in the orthologous murine C1qtnf5 gene by site-directed mutagenesis and homologous recombination into mouse embryonic stem cells. Biochemical, immunological, electron microscopic, fundus autofluorescence, electroretinography and laser photocoagulation analyses were used to characterise the mouse model. Heterozygous and homozygous knock-in mice showed no significant abnormality in any of the above measures at time points up to 2 years. This result contrasts with another C1qtnf5 Ser163Arg knock-in mouse which showed most of the features of L-ORMD but differed in genetic background and targeting construct.

  6. In vivo mutagenesis reveals that OriL is essential for mitochondrial DNA replication

    Science.gov (United States)

    Wanrooij, Sjoerd; Miralles Fusté, Javier; Stewart, James B; Wanrooij, Paulina H; Samuelsson, Tore; Larsson, Nils-Göran; Gustafsson, Claes M; Falkenberg, Maria

    2012-01-01

    The mechanisms of mitochondrial DNA replication have been hotly debated for a decade. The strand-displacement model states that lagging-strand DNA synthesis is initiated from the origin of light-strand DNA replication (OriL), whereas the strand-coupled model implies that OriL is dispensable. Mammalian mitochondria cannot be transfected and the requirements of OriL in vivo have therefore not been addressed. We here use in vivo saturation mutagenesis to demonstrate that OriL is essential for mtDNA maintenance in the mouse. Biochemical and bioinformatic analyses show that OriL is functionally conserved in vertebrates. Our findings strongly support the strand-displacement model for mtDNA replication. PMID:23090476

  7. 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.

  8. Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals

    Directory of Open Access Journals (Sweden)

    van Roekel Henk S

    2008-10-01

    Full Text Available Abstract Background The laboratory rat (Rattus norvegicus is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive. Results As ENU-induced DNA damage is normally recognized by the mismatch repair (MMR system, we hypothesized that the effectiveness of the target-selected mutagenesis approach could be improved by using a MMR-deficient genetic background. Indeed, Msh6 knockout rats were found to be more sensitive to ENU treatment and the germ line mutation rate was boosted more than two-fold to 1 mutation per 585 kb. In addition, the molecular mutation spectrum was found to be changed in favor of generating knockout-type alleles by ~20%, resulting in an overall increase in efficiency of ~2.5 fold. The improved effectiveness was demonstrated by high throughput mutation discovery in 70 Mb of sequence in a set of only 310 mutant F1 rats. This resulted in the identification of 89 mutations of which four introduced a premature stopcodon and 64 resulted in amino acid changes. Conclusion Taken together, we show that the use of a MMR-deficient background considerably improves ENU-driven target-selected mutagenesis in the rat, thereby reducing animal use as well as screening costs. The use of a mismatch repair-deficient genetic background for improving mutagenesis and target-selected knockout efficiency is in principle applicable to any organism of interest.

  9. Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals

    Science.gov (United States)

    van Boxtel, Ruben; Toonen, Pim W; Verheul, Mark; van Roekel, Henk S; Nijman, Isaac J; Guryev, Victor; Cuppen, Edwin

    2008-01-01

    Background The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive. Results As ENU-induced DNA damage is normally recognized by the mismatch repair (MMR) system, we hypothesized that the effectiveness of the target-selected mutagenesis approach could be improved by using a MMR-deficient genetic background. Indeed, Msh6 knockout rats were found to be more sensitive to ENU treatment and the germ line mutation rate was boosted more than two-fold to 1 mutation per 585 kb. In addition, the molecular mutation spectrum was found to be changed in favor of generating knockout-type alleles by ~20%, resulting in an overall increase in efficiency of ~2.5 fold. The improved effectiveness was demonstrated by high throughput mutation discovery in 70 Mb of sequence in a set of only 310 mutant F1 rats. This resulted in the identification of 89 mutations of which four introduced a premature stopcodon and 64 resulted in amino acid changes. Conclusion Taken together, we show that the use of a MMR-deficient background considerably improves ENU-driven target-selected mutagenesis in the rat, thereby reducing animal use as well as screening costs. The use of a mismatch repair-deficient genetic background for improving mutagenesis and target-selected knockout efficiency is in principle applicable to any organism of interest. PMID:18840264

  10. Pyrosequencing: applicability for studying DNA damage-induced mutagenesis.

    Science.gov (United States)

    Minko, Irina G; Earley, Lauriel F; Larlee, Kimberly E; Lin, Ying-Chih; Lloyd, R Stephen

    2014-10-01

    Site-specifically modified DNAs are routinely used in the study of DNA damage-induced mutagenesis. These analyses involve the creation of DNA vectors containing a lesion at a pre-determined position, DNA replication, and detection of mutations at the target site. The final step has previously required the isolation of individual DNA clones, hybridization with radioactively labeled probes, and verification of mutations by Sanger sequencing. In the search for an alternative procedure that would allow direct quantification of sequence variants in a mixed population of DNA molecules, we evaluated the applicability of pyrosequencing to site-specific mutagenesis assays. The progeny DNAs were analyzed that originated from replication of N(6) -(deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG)-containing vectors in primate cells, with the lesion being positioned in the 5'-GCNGG-3' sequence context. Pyrosequencing detected ∼8% G to T transversions and ∼3.5% G to A transitions, a result that was in excellent agreement with frequencies previously measured by the standard procedure (Earley LF et al. [2013]: Chem Res Toxicol 26:1108-1114). However, ∼3.5% G to C transversions and ∼2.0% deletions could not be detected by pyrosequencing. Consistent with these observations, the sensitivity of pyrosequencing for measuring the single deoxynucleotide variants differed depending on the deoxynucleotide identity, and in the given sequence contexts, was determined to be ∼1-2% for A and T and ∼5% for C. Pyrosequencing of other DNA isolates that were obtained following replication of MeFapy-dG-containing vectors in primate cells or Escherichia coli, identified several additional limitations. Collectively, our data demonstrated that pyrosequencing can be used for studying DNA damage-induced mutagenesis as an effective complementary experimental approach to current protocols.

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

    OpenAIRE

    Hämäläinen, Riikka H.; Ahlqvist, Kati J.; Ellonen, Pekka; Lepistö, Maija; Logan, Angela; Otonkoski, Timo; Murphy, Michael P.; Suomalainen, Anu

    2015-01-01

    Summary 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 associate...

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

    Science.gov (United States)

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

    2007-10-01

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

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

    Science.gov (United States)

    Jang, Chul Ho; Piao, Yu Lan; Huang, Xiaoqin; Yoon, Eun Jeong; Park, So Hee; Lee, Kyoung; Zhan, Chang-Guo; Cho, Hoon

    2016-01-01

    Alginate is known to prevent elimination of Pseudomonas aeruginosa biofilms. Alginate lyase (AlgL) might therefore facilitate treatment of Pseudomonas aeruginosa-infected cystic fibrosis patients. However, the catalytic activity of wild-type AlgL is not sufficiently high. Therefore, molecular modeling and site-directed mutagenesis of AlgL might assist in enzyme engineering for therapeutic development. AlgL, isolated from Azotobacter vinelandii, catalyzes depolymerization of alginate via a β-elimination reaction. AlgL was modeled based on the crystal structure template of Sphingomonas AlgL species A1-III. Based on this computational analysis, AlgL was subjected to site-directed mutagenesis to improve its catalytic activity. The kcat/Km of the K194E mutant showed a nearly 5-fold increase against the acetylated alginate substrate, as compared to the wild-type. Double and triple mutants (K194E/K245D, K245D/K319A, K194E/K245D/E312D, and K194E/K245D/K319A) were also prepared. The most potent mutant was observed to be K194E/K245D/K319A, which has a 10-fold improved kcat value (against acetylated alginate) compared to the wild-type enzyme. The antibiofilm effect of both AlgL forms was identified in combination with piperacillin/tazobactam (PT) and the disruption effect was significantly higher in mutant AlgL combined with PT than wild-type AlgL. However, for both the wild-type and K194E/K245D/K319A mutant, the use of the AlgL enzyme alone did not show significant antibiofilm effect. PMID:27253324

  14. Prediction of Local Quality of Protein Structure Models Considering Spatial Neighbors in Graphical Models

    Science.gov (United States)

    Shin, Woong-Hee; Kang, Xuejiao; Zhang, Jian; Kihara, Daisuke

    2017-01-01

    Protein tertiary structure prediction methods have matured in recent years. However, some proteins defy accurate prediction due to factors such as inadequate template structures. While existing model quality assessment methods predict global model quality relatively well, there is substantial room for improvement in local quality assessment, i.e. assessment of the error at each residue position in a model. Local quality is a very important information for practical applications of structure models such as interpreting/designing site-directed mutagenesis of proteins. We have developed a novel local quality assessment method for protein tertiary structure models. The method, named Graph-based Model Quality assessment method (GMQ), explicitly considers the predicted quality of spatially neighboring residues using a graph representation of a query protein structure model. GMQ uses conditional random field as its core of the algorithm, and performs a binary prediction of the quality of each residue in a model, indicating if a residue position is likely to be within an error cutoff or not. The accuracy of GMQ was improved by considering larger graphs to include quality information of more surrounding residues. Moreover, we found that using different edge weights in graphs reflecting different secondary structures further improves the accuracy. GMQ showed competitive performance on a benchmark for quality assessment of structure models from the Critical Assessment of Techniques for Protein Structure Prediction (CASP). PMID:28074879

  15. Homology-based modeling of the Erwinia amylovora type III secretion chaperone DspF used to identify amino acids required for virulence and interaction with the effector DspE.

    Science.gov (United States)

    Triplett, Lindsay R; Wedemeyer, William J; Sundin, George W

    2010-09-01

    The structure of DspF, a type III secretion system (T3SS) chaperone required for virulence of the fruit tree pathogen Erwinia amylovora, was modeled based on predicted structural homology to characterized T3SS chaperones. This model guided the selection of 11 amino acid residues that were individually mutated to alanine via site-directed mutagenesis. Each mutant was assessed for its effect on virulence complementation, dimerization and interaction with the N-terminal chaperone-binding site of DspE. Four amino acid residues were identified that did not complement the virulence defect of a dspF knockout mutant, and three of these residues were required for interaction with the N-terminus of DspE. This study supports the significance of the predicted beta-sheet helix-binding groove in DspF chaperone function.

  16. Transcription-associated mutagenesis increases protein sequence diversity more effectively than does random mutagenesis in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Hyunchul Kim

    Full Text Available BACKGROUND: During transcription, the nontranscribed DNA strand becomes single-stranded DNA (ssDNA, which can form secondary structures. Unpaired bases in the ssDNA are less protected from mutagens and hence experience more mutations than do paired bases. These mutations are called transcription-associated mutations. Transcription-associated mutagenesis is increased under stress and depends on the DNA sequence. Therefore, selection might significantly influence protein-coding sequences in terms of the transcription-associated mutability per transcription event under stress to improve the survival of Escherichia coli. METHODOLOGY/PRINCIPAL FINDINGS: The mutability index (MI was developed by Wright et al. to estimate the relative transcription-associated mutability of bases per transcription event. Using the most stable fold of each ssDNA that have an average length n, MI was defined as (the number of folds in which the base is unpaired/nx(highest -DeltaG of all n folds in which the base is unpaired, where DeltaG is the free energy. The MI values show a significant correlation with mutation data under stress but not with spontaneous mutations in E. coli. Protein sequence diversity is preferred under stress but not under favorable conditions. Therefore, we evaluated the selection pressure on MI in terms of the protein sequence diversity for all the protein-coding sequences in E. coli. The distributions of the MI values were lower at bases that could be substituted with each of the other three bases without affecting the amino acid sequence than at bases that could not be so substituted. Start codons had lower distributions of MI values than did nonstart codons. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the majority of protein-coding sequences have evolved to promote protein sequence diversity and to reduce gene knockout under stress. Consequently, transcription-associated mutagenesis increases protein sequence diversity more effectively

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

    Science.gov (United States)

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

    2016-01-01

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

  18. 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.

  19. Heritable site-specific mutagenesis using TALENs in maize.

    Science.gov (United States)

    Char, Si Nian; Unger-Wallace, Erica; Frame, Bronwyn; Briggs, Sarah A; Main, Marcy; Spalding, Martin H; Vollbrecht, Erik; Wang, Kan; Yang, Bing

    2015-09-01

    Transcription activator-like effector nuclease (TALEN) technology has been utilized widely for targeted gene mutagenesis, especially for gene inactivation, in many organisms, including agriculturally important plants such as rice, wheat, tomato and barley. This report describes application of this technology to generate heritable genome modifications in maize. TALENs were employed to generate stable, heritable mutations at the maize glossy2 (gl2) locus. Transgenic lines containing mono- or di-allelic mutations were obtained from the maize genotype Hi-II at a frequency of about 10% (nine mutated events in 91 transgenic events). In addition, three of the novel alleles were tested for function in progeny seedlings, where they were able to confer the glossy phenotype. In a majority of the events, the integrated TALEN T-DNA segregated independently from the new loss of function alleles, producing mutated null-segregant progeny in T1 generation. Our results demonstrate that TALENs are an effective tool for genome mutagenesis in maize, empowering the discovery of gene function and the development of trait improvement.

  20. 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.

  1. 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.

  2. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

  4. Role of DNA repair in ethyl methanesulfonate-induced mutagenesis in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Prakash, L.; Higgins, D.

    1982-01-01

    Ethyl methanesulfonate (EMS)-induced reversion of two sites which revert preferentially by GC to AT transitions, cycl-131 and cycl-115, has been examined in readiation sensitive, rad, mutants of yeast belonging to the rad52 epistasis group. The rad50, rad 51, rad52, rad54 and rad56 mutants showed reduced reversion of both tester sites when stationary phase diploid cells were treated with EMS. No correlation was found between EMS-induced reversion and EMS-induced homologous mitotic intragenic recombination. Survival of rad6 rad52 double mutants following EMS treatment indicates that there is one epistasis group for the repair of EMS-induced lethal damage in yeast. A model involving misrepair mutagenesis of specific lesions is proposed to account for the experimental results.

  5. Establishment of a Counter-selectable Markerless Mutagenesis System in Veillonella atypica

    Science.gov (United States)

    Zhou, Peng; Li, Xiaoli; Qi, Fengxia

    2015-01-01

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

  6. 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 putat

  7. Efficient targeted mutagenesis in medaka using custom-designed transcription activator-like effector nucleases.

    Science.gov (United States)

    Ansai, Satoshi; Sakuma, Tetsushi; Yamamoto, Takashi; Ariga, Hiroyoshi; Uemura, Norihito; Takahashi, Ryosuke; Kinoshita, Masato

    2013-03-01

    Transcription activator-like effector nucleases (TALENs) have become powerful tools for targeted genome editing. Here we demonstrate efficient targeted mutagenesis in medaka (Oryzias latipes), which serves as an excellent vertebrate model for genetics and genomics. We designed and constructed a pair of TALENs targeting the medaka DJ-1 gene, a homolog of human DJ-1 (PARK7). These TALENs induced a number of insertions and deletions in the injected embryos with extremely high efficiency. This induction of mutations occurred in a dose-dependent manner. All screened G0 fish injected with the TALENs transmitted the TALEN-induced mutations to the next generation with high efficiency (44-100%). We also confirmed that these TALENs induced site-specific mutations because none of the mutations were found at potential off-target sites. In addition, the DJ-1 protein was lost in DJ-1(Δ7/Δ7) fish that carried a TALEN-induced frameshift mutation in both alleles. We also investigated the effect of the N- and C-terminal regions of the transcription activator-like (TAL) effector domain on the gene-disrupting activity of DJ1-TALENs and found that 287 amino acids at the N terminus and 63 amino acids at the C terminus of the TAL domain exhibited the highest disrupting activity in the injected embryos. Our results suggest that TALENs enable us to rapidly and efficiently establish knockout medaka strains. This is the first report of targeted mutagenesis in medaka using TALENs. The TALEN technology will expand the potential of medaka as a model system for genetics and genomics.

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

    Directory of Open Access Journals (Sweden)

    Maura McGrail

    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.

  9. 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...... sequences were reconstructed as an oligonucleotide cassette. Based upon the ability of the mutants to induce focal transformation of NIH 3T3 cells, a range of phenotypes from virtually full activity to none (null mutants) was seen. Three classes of codons were present in this segment: one which could...

  10. Radiosensitivity Parameters For Lethal Mutagenesis In Caenorhabditis Elegans

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Mouse Mutagenesis Using N-Ethyl-N-Nitrosourea (ENU).

    Science.gov (United States)

    Salinger, Andrew P; Justice, Monica J

    2008-04-01

    INTRODUCTIONThis protocol describes chemical mutagenesis of male mice using N-ethyl-N-nitrosourea (ENU), which is the most efficient method for obtaining mouse mutations in phenotype-driven screens. A fractionated dose of ENU, an alkylating agent, can produce a mutation rate as high as 1.5 × 10(-3) in male mouse spermatogonial stem cells. Treatment with ENU produces point mutations that provide a unique mutant resource: They reflect the consequences of single gene changes independent of position effects, provide a fine structure dissection of protein function, display a range of mutant effects from complete or partial loss of function to exaggerated function, and discover gene functions in an unbiased manner. After treatment with ENU, mice are mated in genetic screens designed to uncover mutations of interest. Screens for dominant, recessive, and modifying mutations can be performed.

  13. 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.

  14. Mechanisms of uv mutagenesis in yeast and E. coli

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence, C.; Christensen, R.; Christensen, J.R.; O' Brien, T.

    1983-01-01

    Experiments investigating ultraviolet light mutagenesis in either bakers' yeast, Saccharomyces cerevisiae, or E. coli have led to the following conclusions. First, cyclobutane pyrimidine dimers cause most mutations in both organisms; pyrimidine adducts, such as PyC, can account at best for only a small proportion. 86 percent of forward mutations induced at the E. coli lacI locus can be abolished by photoreactivation under conditions which do not alter the level of recA induction. About 75 percent of the forward mutations induced at the CAN1 locus of yeast could be removed by photoreactivation, a value that lies within the range observed previously for the reversion of CYC1 alleles (60 percent - 97 percent). Second, about 10 percent of the lacI forward mutations are untargeted, a smaller fraction than found previously for cycl-91 reversion in yeast. It is not yet clear whether the two species are really different in this respect, of whether the cycl-91 reversion site is a typical of the yeast genome at large. Third, analysis of reversion frequencies of 20 mutant alleles suggests that about 10 to 25 percent of all replication errors produced by mutagenic mechanisms in uv-irradiated yeast involve additions or deletions of base-pairs, indicating that error-prone repair does not just produce substitutions. Last, the REV1 locus in yeast is concerned with the induction of frameshift mutations at some, but not all, genetic sites, just as found previously for substitution mutations. The function of the REV3 gene is more widely, though not universally, required while the function of the RAD6 gene, like that of the recA locus in E. coli, appears to be necessary for all kinds of uv mutagenesis. E coli genes comparable to REV1 and REV3 have not yet been described; conversely, there does not yet appear to be a yeast equivalent of umuC.

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

    Science.gov (United States)

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

    2015-01-01

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

  16. Genetic, biochemical characterization and mutagenesis of the chromosomal class A β-lactamase of Raoultella (formerly Klebsiella) terrigena.

    Science.gov (United States)

    Walckenaer, E; Delmas, J; Leflon-Guibout, V; Bonnet, R; Nicolas-Chanoine, M-H

    2015-09-01

    Chromosomal class A β-lactamases have been characterized in Raoultella ornithinolytica and Raoultella planticola. The purpose of this study was to characterize that of Raoultella terrigena. The blaTER-1 gene of R. terrigena strain ATCC33257(T) was cloned (pACter-1) and sequenced. It was then used to detect the bla gene of strains BM 85 01 095 and SB2796. The hypermutable Escherichia coli strain AB1157 mutS::Tn10 was transformed with pACter-1 and mutants growing on plates containing>2mg/L ceftazidime were studied. Notably, the impact of mutations only observed in the promoter region on β-lactam resistance was assessed by site-directed mutagenesis experiments. R. terrigena strains ATCC33257(T) and BM 85 01 095 had the same bla gene and deduced protein (TER-1) whereas there were 3 substitutions in those of strain SB2796 (TER-2). Class A β-lactamases TER showed 78%, 69.9% and 38.7% identity with PLA or ORN, TEM-1 and KOXY, respectively. Compared with TEM-1, TER-1 and TER-2 showed 2 particular substitutions, Leu75Pro and Glu240Asn demonstrated to be involved in the inherent β-lactam resistance profile of R. terrigena. TER-1 (pI of 7.6) had a high activity against penicillin G and a significantly low one against amoxicillin. Substitution G/T observed in the -35 region of the blaTER gene harbored by strains growing in the presence of≥2mg/L ceftazidime was shown to be responsible for this growth. TER is a new class A β-lactamase belonging to functional group 2b. Copyright © 2015. Published by Elsevier SAS.

  17. Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris.

    Science.gov (United States)

    Chang, S W; Shieh, C J; Lee, G C; Shaw, J F

    2005-04-01

    Candida rugosa lipase, a significant catalyst, had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications. Several isozymes encoded by the lip gene family, namely lip1 to lip7, possess distinct thermal stability and substrate specificity, among which the recombinant LIP1 showed a distinguished catalytic characterization. In this study, we utilized PCR to remove an unnecessary linker of pGAPZalphaC vector and used overlap extension PCR-based multiple site-directed mutagenesis to convert the 19 non-universal CTG-serine codons into universal TCT-serine codons and successfully express a highly active recombinant C. rugosa LIP1 in the Pichia expression system. Response surface methodology and 4-factor-5-level central composite rotatable design were adopted to evaluate the effects of growth parameters, such as temperature (21.6-38.4 degrees C), glucose concentration (0.3-3.7%), yeast extract (0.16-1.84%), and pH (5.3-8.7) on the lipolytic activity of LIP1 and biomass of P. pastoris. Based on ridge max analysis, the optimum LIP1 production conditions were temperature, 24.1 degrees C; glucose concentration, 2.6%; yeast extract, 1.4%; and pH 7.6. The predicted value of lipolytic activity was 246.9+/-39.7 U/ml, and the actual value was 253.3+/-18.8 U/ml. The lipolytic activity of the recombinant LIP1 resulting from the present work is twofold higher than that achieved by a methanol induction system.

  18. Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts.

    Science.gov (United States)

    Andersson, Mariette; Turesson, Helle; Nicolia, Alessandro; Fält, Ann-Sofie; Samuelsson, Mathias; Hofvander, Per

    2017-01-01

    Altered starch quality with full knockout of GBSS gene function in potato was achieved using CRISPR-Cas9 technology, through transient transfection and regeneration from isolated protoplasts. Site-directed mutagenesis (SDM) has shown great progress in introducing precisely targeted mutations. Engineered CRISPR-Cas9 has received increased focus compared to other SDM techniques, since the method is easily adapted to different targets. Here, we demonstrate that transient application of CRISPR-Cas9-mediated genome editing in protoplasts of tetraploid potato (Solanum tuberosum) yielded mutations in all four alleles in a single transfection, in up to 2 % of regenerated lines. Three different regions of the gene encoding granule-bound starch synthase (GBSS) were targeted under different experimental setups, resulting in mutations in at least one allele in 2-12 % of regenerated shoots, with multiple alleles mutated in up to 67 % of confirmed mutated lines. Most mutations resulted in small indels of 1-10 bp, but also vector DNA inserts of 34-236 bp were found in 10 % of analysed lines. No mutations were found in an allele diverging one bp from a used guide sequence, verifying similar results found in other plants that high homology between guide sequence and target region near the protospacer adjacent motif (PAM) site is essential. To meet the challenge of screening large numbers of lines, a PCR-based high-resolution fragment analysis method (HRFA) was used, enabling identification of multiple mutated alleles with a resolution limit of 1 bp. Full knockout of GBSS enzyme activity was confirmed in four-allele mutated lines by phenotypic studies of starch. One remaining wild-type (WT) allele was shown sufficient to maintain enough GBSS enzyme activity to produce significant amounts of amylose.

  19. Molecular model of the neural dopamine transporter

    Science.gov (United States)

    Ravna, Aina Westrheim; Sylte, Ingebrigt; Dahl, Svein G.

    2003-05-01

    The dopamine transporter (DAT) regulates the action of dopamine by reuptake of the neurotransmitter into presynaptic neurons, and is the main molecular target of amphetamines and cocaine. DAT and the Na+/H+ antiporter (NhaA) are secondary transporter proteins that carry small molecules across a cell membrane against a concentration gradient, using ion gradients as energy source. A 3-dimensional projection map of the E. coli NhaA has confirmed a topology of 12 membrane spanning domains, and was previously used to construct a 3-dimensional NhaA model with 12 trans-membrane α-helices (TMHs). The NhaA model, and site directed mutagenesis data on DAT, were used to construct a detailed 3-dimensional DAT model using interactive molecular graphics and empiric force field calculations. The model proposes a dopamine transport mechanism involving TMHs 1, 3, 4, 5, 7 and 11. Asp79, Tyr252 and Tyr274 were the primary cocaine binding residues. Binding of cocaine or its analogue, (-)-2β-carbomethoxy-3β-(4-fluorophenyl)tropane (CFT), seemed to lock the transporter in an inactive state, and thus inhibit dopamine transport. The present model may be used to design further experimental studies of the molecular structure and mechanisms of DAT and other secondary transporter proteins.

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

    Directory of Open Access Journals (Sweden)

    Omid Zarei

    2017-04-01

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

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

    Science.gov (United States)

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

    2009-10-01

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

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

    Science.gov (United States)

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

    2001-03-01

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

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

    Science.gov (United States)

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

    2014-05-20

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

  4. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

    Science.gov (United States)

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

    2015-12-18

    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

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

    Directory of Open Access Journals (Sweden)

    Asenjo Juan A

    2011-04-01

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

  6. Structural characteristics of the hydrophobic patch of azurin and its interaction with p53: a site-directed spin labeling study

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for monitoring protein structure, dynamics and conformational changes. In this study, the domain-specific properties of azurin and its interaction with p53 were studied using this technique. Mutations of six residues, that are located in the hydrophobic patch of azurin, were prepared and spin labeled. Spectra of the six azurin mutants in solution showed that spin labeled residues 45 and 63 are in a very restricted environment, residues 59 and 65 are in a spacious environment and have free movement, and residues 49 and 51 are located in a relatively closed pocket. Polarity experiments confirmed these results. The changes observed in the spectra of spin labeled azurin upon interaction with p53 indicate that the hydrophobic patch is involved in this interaction. Our results provide valuable insight into the topographic structure of the hydrophobic domain of azurin, as well as direct evidence of its interaction with p53 in solution via the hydrophobic patch. Cytotoxicity studies of azurin mutants showed that residues along the hydrophobic patch are important for its cytotoxicity.

  7. 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.

  8. Bromopyruvate, an active site-directed inactivator of E. coli 2-keto-4-hydroxyglutarate(KHG) aldolase, modifies glutamic acid residue-45

    Energy Technology Data Exchange (ETDEWEB)

    Vlahos, C.J.; Dekker, E.E.

    1987-05-01

    E. coli KHG-aldolase (2-keto-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate), a novel trimeric Class I aldolase, requires one active-site lysine residue (Lys 133)/subunit for Schiff-base formation as well as one arginine residue (Arg 49)/subunit for catalytic activity. The substrate analog, 3-bromopyruvate (BRPY), causes a time- and concentration-dependent loss of KHG-aldolase activity. This inactivation is regarded as active site-directed since: (a) BRPY modification results in complete loss of enzymatic activity; (b) saturation kinetics are exhibited, suggesting that a reversible complex is formed between the aldolase and BRPY prior to the rate-limiting inactivation step; (c) over 90% of the initial aldolase activity is protected by either substrate, pyruvate or KHG; (d) 1.1 mol of /sup 14/C-BRPY is bound/enzyme subunit. Peptide isolation and sequencing show that the incorporated radioactivity is associated with residue Glu-45. Denaturation of the enzyme with guanidine x HCl following treatment with excess /sup 14/C-BRPY allows for the incorporation of carbon-14 at Cys-159 and Cys-180 as well. The presence of pyruvate protects Glu-45 from being esterified but does not prevent the alkylation of the two cysteine residues. These results suggest that Glu-45 is essential for the catalytic activity of E. coli KHG-aldolase, most likely functioning as the active-site amphoteric proton donor/acceptor moiety that is involved in the overall mechanism of the reaction catalyzed by this enzyme.

  9. Site-directed RNA editing by adenosine deaminase acting on RNA (ADAR1) for correction of the genetic code in gene therapy.

    Science.gov (United States)

    Azad, T A; Bhakta, S; Tsukahara, T

    2017-10-06

    Site-directed RNA editing is an important technique for correcting gene sequences and ultimately tuning protein function. In this study, we engineered the deaminase domain of adenosine deaminase acting on RNA (ADAR1) and the MS2 system to target specific adenosines, with the goal of correcting G-to-A mutations at the RNA level. For this purpose, the ADAR1 deaminase domain was fused downstream of the RNA-binding protein MS2, which has affinity for the MS2 RNA. To direct editing to specific targets, we designed guide RNAs complementary to target RNAs. The guide RNAs directed the ADAR1 deaminase to the desired editing site, where it converted adenosine to inosine. To provide proof of principle, we used an allele of EGFP bearing a mutation at the 58th amino acid (TGG), encoding Trp, into an amber (TAG) or ochre (TAA) stop codon. In HEK-293 cells, our system could convert stop codons to read-through codons, thereby turning on fluorescence. We confirmed the specificity of editing at the DNA level by restriction fragment length polymorphism (RFLP) analysis and sequencing, and at the protein level by western blotting. The editing efficiency of this enzyme system was ~5%. We believe that this system could be used to treat genetic diseases resulting from G-to-A point mutations.Gene Therapy accepted article preview online, 06 October 2017. doi:10.1038/gt.2017.90.

  10. 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.

  11. ldentification and Mutagenesis of Lactic Acid Bacteria from Chinese Sauerkraut

    Institute of Scientific and Technical Information of China (English)

    Yajing CHAl; Hao SHl; Ri NA

    2015-01-01

    ln order to analyze the fermentation properties of lactic acid bacteria in Chinese sauerkraut and to improve acid production, 21 samples of Chinese sauerkraut from lnner Mongolia and Northeast China were col ected and isolated with a Man-Rogosa-Sharpe (MRS) culture. Sixteen strains of lactic acid bacteria were identified by combining both phenotype and genotype methods. After activation, the 16 strains were inoculated into the MRS medium with a concentration of 4%and then incubated at 37 ℃. The pH and the absorbance of the culture were mea-sured. The activated strains were then mutagenized in a field of 4 KV/cm mutation, with dosages administered within 20 minutes and 30 minutes, respectively. The variation curves of the pH and the absorbance of the culture were determined. The experimental results showed that the lactic acid bacteria isolated from the soup were identified as Lactobacil us and the acid production of the bacteria was signifi-cantly improved by the mutagenesis of the corona electric field.

  12. Ultraviolet mutagenesis and inducible DNA repair in Caulobacter crescentus

    Energy Technology Data Exchange (ETDEWEB)

    Bender, R.A.

    1984-11-19

    The ability to reactivate ultraviolet (UV) damaged phage phiCbK (W-reactivation) is induced by UV irradiation of Caulobacter crescentus cells. Induction of W-reactivation potential is specific for phage phiCbK, requires protein synthesis, and is greatly reduced in the presence of the rec-526 mutation. The induction signal generated by UV irradiation is transient, lasting about 1 1/2 - 2 h at 30/sup 0/C; if chloramphenicol is present during early times after UV irradiation, induction of W-reactivation does not occur. Induction is maximal when cells are exposed to 5-10 J/m/sup 2/ of UV, a dose that also results in considerable mutagenesis of the cells. Taken together, these observations demonstrate the existence of a UV inducible, protein synthesis requiring, transiently signalled, rec-requiring DNA repair system analogous to W-reactivation in Escherichia coli. In addition, C. crescentus also has an efficient photoreactivation system that reverses UV damage in the presence of strong visible light.

  13. Radio frequency electromagnetic fields: cancer, mutagenesis, and genotoxicity.

    Science.gov (United States)

    Heynick, Louis N; Johnston, Sheila A; Mason, Patrick A

    2003-01-01

    We present critiques of epidemiologic studies and experimental investigations, published mostly in peer-reviewed journals, on cancer and related effects from exposure to nonionizing electromagnetic fields in the nominal frequency range of 3 kHz to 300 GHz of interest to Subcommittee 4 (SC4) of the International Committee on Electromagnetic Safety (ICES). The major topics discussed are presented under the headings Epidemiologic and Other Findings on Human Exposure, Mammals Exposed In Vivo, Mammalian Live Tissues and Cell Preparations Exposed In Vitro, and Mutagenesis and Genotoxicity in Microorganisms and Fruit Flies. Under each major topic, we present minireviews of papers on various specific endpoints investigated. The section on Epidemiologic and Other Findings on Human Exposure is divided into two subsections, the first on possible carcinogenic effects of exposure from emitters not in physical contact with the populations studied, for example, transmitting antennas and other devices. Discussed in the second subsection are studies of postulated carcinogenic effects from use of mobile phones, with prominence given to brain tumors from use of cellular and cordless telephones in direct physical contact with an ear of each subject. In both subsections, some investigations yielded positive findings, others had negative findings, including papers directed toward experimentally verifying positive findings, and both were reported in a few instances. Further research on various important aspects may resolve such differences. Overall, however, the preponderance of published epidemiologic and experimental findings do not support the supposition that in vivo or in vitro exposures to such fields are carcinogenic.

  14. 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

  15. Retroviral Vectors for Analysis of Viral Mutagenesis and Recombination

    Directory of Open Access Journals (Sweden)

    Jonathan M.O. Rawson

    2014-09-01

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

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

    Science.gov (United States)

    Rawson, Jonathan M O; Mansky, Louis M

    2014-09-24

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

  17. Insertion and deletion mutagenesis of the human cytomegalovirus genome

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-10-01

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

  18. Targeted mutagenesis using CRISPR/Cas system in medaka

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    Satoshi Ansai

    2014-04-01

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

  19. Site Saturation Mutagenesis Applications on Candida methylica Formate Dehydrogenase

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    Gülşah P. Özgün

    2016-01-01

    Full Text Available In NADH regeneration, Candida methylica formate dehydrogenase (cmFDH is a highly significant enzyme in pharmaceutical industry. In this work, site saturation mutagenesis (SSM which is a combination of both rational design and directed evolution approaches is applied to alter the coenzyme specificity of NAD+-dependent cmFDH from NAD+ to NADP+ and increase its thermostability. For this aim, two separate libraries are constructed for screening a change in coenzyme specificity and an increase in thermostability. To alter the coenzyme specificity, in the coenzyme binding domain, positions at 195, 196, and 197 are subjected to two rounds of SSM and screening which enabled the identification of two double mutants D195S/Q197T and D195S/Y196L. These mutants increase the overall catalytic efficiency of NAD+ to 5.6×104-fold and 5×104-fold value, respectively. To increase the thermostability of cmFDH, the conserved residue at position 1 in the catalytic domain of cmFDH is subjected to SSM. The thermodynamic and kinetic results suggest that 8 mutations on the first residue can be tolerated. Among all mutants, M1L has the best residual activity after incubation at 60°C with 17%. These studies emphasize that SSM is an efficient method for creating “smarter libraries” for improving the properties of cmFDH.

  20. Reverse genetics through random mutagenesis in Histoplasma capsulatum

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    Rappleye Chad A

    2009-11-01

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

  1. Mutagenesis-mediated virus extinction: virus-dependent effect of viral load on sensitivity to lethal defection.

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    Héctor Moreno

    Full Text Available BACKGROUND: Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication, and it is currently under investigation as a potential new antiviral strategy. Viral load and virus fitness are known to influence virus extinction. Here we examine the effect or the multiplicity of infection (MOI on progeny production of several RNA viruses under enhanced mutagenesis. RESULTS: The effect of the mutagenic base analogue 5-fluorouracil (FU on the replication of the arenavirus lymphocytic choriomeningitis virus (LCMV can result either in inhibition of progeny production and virus extinction in infections carried out at low multiplicity of infection (MOI, or in a moderate titer decrease without extinction at high MOI. The effect of the MOI is similar for LCMV and vesicular stomatitis virus (VSV, but minimal or absent for the picornaviruses foot-and-mouth disease virus (FMDV and encephalomyocarditis virus (EMCV. The increase in mutation frequency and Shannon entropy (mutant spectrum complexity as a result of virus passage in the presence of FU was more accentuated at low MOI for LCMV and VSV, and at high MOI for FMDV and EMCV. We present an extension of the lethal defection model that agrees with the experimental results. CONCLUSIONS: (i Low infecting load favoured the extinction of negative strand viruses, LCMV or VSV, with an increase of mutant spectrum complexity. (ii This behaviour is not observed in RNA positive strand viruses, FMDV or EMCV. (iii The accumulation of defector genomes may underlie the MOI-dependent behaviour. (iv LCMV coinfections are allowed but superinfection is strongly restricted in BHK-21 cells. (v The dissimilar effects of the MOI on the efficiency of mutagenic-based extinction of different RNA viruses can have implications for the design of antiviral protocols based on lethal mutagenesis, presently under development.

  2. Site-directed immobilization of antibody using EDC-NHS-activated protein A on a bimetallic-based surface plasmon resonance chip

    Science.gov (United States)

    Sohn, Young-Soo; Lee, Yeon Kyung

    2014-05-01

    The characteristics of a waveguide-coupled bimetallic surface plasmon resonance (WcBiM SPR) sensor using (3-dimethylaminopropyl)-3-ethylcarbodiimide(EDC)-N-hydroxysuccinimide(NHS)-activated protein A was investigated, and the detection of IgG using the EDC-NHS-activated protein A was studied in comparison with protein A and a self-assembled monolayer (SAM). The WcBiM sensor, which has a narrower full width at half maximum (FWHM) and a steeper slope, was selected since it leads to a larger change in the reflectance in the intensity detection mode. A preparation of the EDC-NHS-activated protein A for site-directed immobilization of antibodies was relative easily compared to the engineered protein G and A. In antigen-antibody interactions, the response to IgG at the concentrations of 50, 100, and 150 ng/ml was investigated. The results showed that the sensitivity of the WcBiM sensor using the EDC-NHS-activated protein A, protein A, and SAM was 0.0185 [%/(ng/ml)], 0.0065 [%/(ng/ml)], and 0.0101 [%/(ng/ml)], respectively. The lowest detectable concentrations of IgG with the EDC-NHS-activated protein A, protein A, and SAM were 4.27, 12.83, and 8.24 ng/ml, respectively. Therefore, the increased sensitivity and lower detection capability of the WcBiM SPR chip with the EDC-NHS-activated protein A suggests that it could be used in early diagnosis where the trace level concentrations of biomolecules should be detected.

  3. Mapping alpha-helical induced folding within the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by site-directed spin-labeling EPR spectroscopy.

    Science.gov (United States)

    Belle, Valérie; Rouger, Sabrina; Costanzo, Stéphanie; Liquière, Elodie; Strancar, Janez; Guigliarelli, Bruno; Fournel, André; Longhi, Sonia

    2008-12-01

    Using site-directed spin-labeling EPR spectroscopy, we mapped the region of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)) that undergoes induced folding. In addition to four spin-labeled N(TAIL) variants (S407C, S488C, L496C, and V517C) (Morin et al. (2006), J Phys Chem 110: 20596-20608), 10 new single-site cysteine variants were designed, purified from E. coli, and spin-labeled. These 14 spin-labeled variants enabled us to map in detail the gain of rigidity of N(TAIL) in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol or the C-terminal domain X (XD) of the viral phosphoprotein. Different regions of N(TAIL) were shown to contribute to a different extent to the binding to XD, while the mobility of the spin labels grafted at positions 407 and 460 was unaffected upon addition of XD; that of the spin labels grafted within the 488-502 and the 505-522 regions was severely and moderately reduced, respectively. Furthermore, EPR experiments in the presence of 30% sucrose allowed us to precisely map to residues 488-502, the N(TAIL) region undergoing alpha-helical folding. The mobility of the 488-502 region was found to be restrained even in the absence of the partner, a behavior that could be accounted for by the existence of a transiently populated folded state. Finally, we show that the restrained motion of the 505-522 region upon binding to XD is due to the alpha-helical transition occurring within the 488-502 region and not to a direct interaction with XD.

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

    Science.gov (United States)

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

    2017-03-15

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

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

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    Olga Fernández-Miragall

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

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

    Science.gov (United States)

    Fernández-Miragall, Olga; Hernández, Carmen

    2011-01-01

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

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

    Science.gov (United States)

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

    2016-04-01

    The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis L-serine/L-threonine exchanger is the best-known prokaryotic paradigm of the mammalian L-amino acid transporter (LAT) family. Unfortunately, SteT's lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest. © 2016 Rodríguez-Banqueri et al.

  8. Radiation mutagenesis from molecular and genetic points of view

    Energy Technology Data Exchange (ETDEWEB)

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

    1993-01-01

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

  9. Radiation mutagenesis from molecular and genetic points of view

    Energy Technology Data Exchange (ETDEWEB)

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

    1993-02-01

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

  10. Generation of Enterobacter sp. YSU auxotrophs using transposon mutagenesis.

    Science.gov (United States)

    Caguiat, Jonathan James

    2014-10-31

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

  11. Probing Structural Dynamics and Topology of the KCNE1 Membrane Protein in Lipid Bilayers via Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy.

    Science.gov (United States)

    Sahu, Indra D; Craig, Andrew F; Dunagan, Megan M; Troxel, Kaylee R; Zhang, Rongfu; Meiberg, Andrew G; Harmon, Corrinne N; McCarrick, Robert M; Kroncke, Brett M; Sanders, Charles R; Lorigan, Gary A

    2015-10-20

    KCNE1 is a single transmembrane protein that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in the genes encoding either protein can result in diseases such as congenital deafness, long QT syndrome, ventricular tachyarrhythmia, syncope, and sudden cardiac death. Despite the biological significance of KCNE1, the structure and dynamic properties of its physiologically relevant native membrane-bound state are not fully understood. In this study, the structural dynamics and topology of KCNE1 in bilayered lipid vesicles was investigated using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A 53-residue nitroxide EPR scan of the KCNE1 protein sequence including all 27 residues of the transmembrane domain (45-71) and 26 residues of the N- and C-termini of KCNE1 in lipid bilayered vesicles was analyzed in terms of nitroxide side-chain motion. Continuous wave-EPR spectral line shape analysis indicated the nitroxide spin label side-chains located in the KCNE1 TMD are less mobile when compared to the extracellular region of KCNE1. The EPR data also revealed that the C-terminus of KCNE1 is more mobile when compared to the N-terminus. EPR power saturation experiments were performed on 41 sites including 18 residues previously proposed to reside in the transmembrane domain (TMD) and 23 residues of the N- and C-termini to determine the topology of KCNE1 with respect to the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) lipid bilayers. The results indicated that the transmembrane domain is indeed buried within the membrane, spanning the width of the lipid bilayer. Power saturation data also revealed that the extracellular region of KCNE1 is solvent-exposed with some of the portions partially or weakly interacting with the membrane surface. These results are consistent with the previously published solution NMR

  12. A site-directed spin-labeling study of ligand-induced conformational change in the ferric enterobactin receptor, FepA.

    Science.gov (United States)

    Liu, J; Rutz, J M; Klebba, P E; Feix, J B

    1994-11-15

    The ferric enterobactin receptor, FepA, is a TonB-dependent gated porin that transports the siderophore ferric enterobactin across the outer membrane of gram-negative bacteria. We have created two site-directed mutants of Escherichia coli FepA, in both cases introducing a cysteine residue into the putative ligand-binding domain. The introduced cysteines were then modified with nitroxide spin labels for structural and dynamic studies using electron spin resonance (ESR) spectroscopy. The mutants were fully functional, as indicated by their ability to grow under iron-limiting conditions, their uptake of [59Fe]enterobactin, and their sensitivity to colicin B. Labeling of the mutant FepA receptors proceeded easily upon incubation with sulfhydryl-specific spin labels, e.g. MTSL, (1-oxy-2,2,5,5-tetramethylpyrrolidin-3-yl)methyl methanethiosulfonate. In contrast, spin labeling of the two native cysteines (Cys486 and Cys493) within wild-type FepA occurred only after treatment with a thiol reducing agent and partial denaturation in urea, suggesting that the native cysteines are disulfide-linked. ESR spectra showed a high degree of motional restriction for all three sites. Continuous wave (CW) saturation studies indicated that one of the mutationally introduced sites (Cys280) was surface-localized as evidenced by its exposure to the aqueous paramagnetic relaxation agent chromium oxalate and its low accessibility to O2. The other (Cys310) apparently occupies a site near the membrane/aqueous interface. The native cysteines occupy a site tightly packed within the protein structure with low accessibility to both CROX and O2. A shift in both conventional and saturation-transfer ESR spectra of MTSL-labeled E280C and E310C (but not MTSL-labeled wild type) FepA was observed upon addition of ferric enterobactin. The ESR spectral shift was dependent on ferric enterobactin concentration and did not occur with siderophores not recognized by FepA. Ferric enterobactin binding did not alter

  13. 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.

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

    Science.gov (United States)

    Su, Fang; Huang, Zongliang; Guo, Yawen; Jiao, Renjie; Zi, Li; Chen, Jianming; Liu, Jiyong

    2016-01-01

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

  15. Heteroatom-substituted analogues of the active-site directed inhibitor estra-1,3,5(10)-trien-17-one-3-sulphamate inhibit estrone sulphatase by a different mechanism.

    Science.gov (United States)

    Woo, L W; Lightowler, M; Purohit, A; Reed, M J; Potter, B V

    1996-01-01

    Estrogens have a pivotal role in the growth and development of hormone-dependent breast cancers. In postmenopausal women, the hydrolysis of the conjugate estrone sulphate (E1S) to estrone (E1) by the enzyme estrone sulphatase is the major source of breast tumour estrogen. Inhibitors of estrone sulphatase should therefore have considerable therapeutic potential for the treatment of hormone-dependent tumours of the breast, either as the sole agent or in conjunction with aromatase inhibitors. Several inhibitors of estrone sulphatase have now been developed of which estra-1,3,5(10)-trien-17-one-3-sulphamate (EMATE) is the most potent and also inhibits the enzyme in a time- and concentration-dependent manner, showing that it acts as an irreversible inhibitor. Analogues of EMATE in which the 3-O-atom is replaced by other heteroatoms (S and N) were synthesized and tested for inhibition against estrone sulphatase. 4-Methoxyphenylsulphamide (1), 4-chlorothiophenyl-S-(N,N-dimethyl)sulphamate (2), estra-1,3,5(10)-trien-17-one-3-sulphamide (3), estra-1,3,5(10)-trien-17-one-3-S-sulphamate (4) and estra-1,3,5(10)-trien-17-one-3-S-(N,N-dimethyl)sulphamate (5) were found to inhibit estrone sulphatase weakly, but none of these compounds appears to behave as a time-dependent inhibitor. A model of the mechanism of enzyme inhibition by EMATE is proposed and we conclude that the sulphamate bridging oxygen atom of EMATE is essential for active site-directed inhibition of estrone sulphatase.

  16. 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.

  17. 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.

  18. 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.

    2017-01-01

    ABSTRACT   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. PMID:28096490

  19. 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.

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    Matthew J. Markert

    2016-04-01

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

  2. Single chain human interleukin 5 and its asymmetric mutagenesis for mapping receptor binding sites.

    Science.gov (United States)

    Li, J; Cook, R; Dede, K; Chaiken, I

    1996-01-26

    Wild type human (h) interleukin 5 (wt IL5) is composed of two identical peptide chains linked by disulfide bonds. A gene encoding a single chain form of hIL5 dimer was constructed by linking the two hIL5 chain coding regions with Gly-Gly linker. Expression of this gene in COS cells yielded a single chain IL5 protein (sc IL5) having biological activity similar to that of wt IL5, as judged by stimulation of human cell proliferation. Single chain and wt IL5 also had similar binding affinity for soluble IL5 receptor alpha chain, the specificity subunit of the IL5 receptor, as measured kinetically with an optical biosensor. The design of functionally active sc IL5 molecule. Such mutagenesis was exemplified by changes at residues Glu-13, Arg-91, Glu-110, and Trp-111. The receptor binding and bioactivity data obtained are consistent with a model in which residues from both IL5 monomers interact with the receptor alpha chain, while the interaction likely is asymmetric due to the intrinsic asymmetry of folded receptor. The results demonstrate a general route to the further mapping of receptor and other binding sites on the surface of human IL5.

  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. Commentary on "tissue-specific mutagenesis by N-butyl-N-(4-hydroxybutyl) nitrosamine as the basis for urothelial cell carcinogenesis." He Z, Kosinska W, Zhao ZL, Wu XR, Guttenplan JB, Department of Basic Science, New York University Dental College, NY, USA.: Mutat Res 2012;742(1-2):92-5 [Epub 2011 Dec 4].

    Science.gov (United States)

    Scherr, Douglas S

    2014-02-01

    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 © 2014 Elsevier Inc. All rights reserved.

  5. Exploring the potential of megaprimer PCR in conjunction with orthogonal array design for mutagenesis library construction.

    Science.gov (United States)

    Tang, Lixia; Zheng, Kai; Liu, Yu; Zheng, Huayu; Wang, Hu; Song, Chunlei; Zhou, Hong

    2013-01-01

    Although megaprimer PCR mutagenesis has been used routinely in protein directed evolution, users sometimes encounter technical hurdles, particularly inefficiency during amplification when large fragments are used or the template is difficult to be amplified. Instead of methodology development, here we simply overcome the limitation by optimizing megaprimer PCR conditions via orthogonal array design of the four PCR components in three levels of each: template, primer, Mg(2+) , and dNTPs. For this, only nine PCRs need to be performed. The strategy (termed as OptiMega) was not only successfully applied for the construction of one multiple-site saturation mutagenesis library of halohydrin dehalogenase HheC, which failed to be constructed previously using the standard QuikChange™ protocol, but also expanded the construction of two high-quality random mutagenesis libraries of HheA and HheC. Most importantly, OptiMega offers a quick and simple way of constructing random mutagenesis libraries by eliminating the ligation step. Our results demonstrated that the OptiMega strategy could greatly strengthen the potential of megaprimer PCR mutagenesis for library construction.

  6. 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.

  7. Running loose or getting lost: how HIV-1 counters and capitalizes on APOBEC3-induced mutagenesis through its Vif protein.

    Science.gov (United States)

    Münk, Carsten; Jensen, Björn-Erik O; Zielonka, Jörg; Häussinger, Dieter; Kamp, Christel

    2012-11-14

    Human immunodeficiency virus-1 (HIV-1) dynamics reflect an intricate balance within the viruses’ host. The virus relies on host replication factors, but must escape or counter its host’s antiviral restriction factors. The interaction between the HIV-1 protein Vif and many cellular restriction factors from the APOBEC3 protein family is a prominent example of this evolutionary arms race. The viral infectivity factor (Vif) protein largely neutralizes APOBEC3 proteins, which can induce in vivo hypermutations in HIV-1 to the extent of lethal mutagenesis, and ensures the production of viable virus particles. HIV-1 also uses the APOBEC3-Vif interaction to modulate its own mutation rate in harsh or variable environments, and it is a model of adaptation in a coevolutionary setting. Both experimental evidence and the substantiation of the underlying dynamics through coevolutionary models are presented as complementary views of a coevolutionary arms race.

  8. Running Loose or Getting Lost: How HIV-1 Counters and Capitalizes on APOBEC3-Induced Mutagenesis through Its Vif Protein

    Directory of Open Access Journals (Sweden)

    Christel Kamp

    2012-11-01

    Full Text Available Human immunodeficiency virus-1 (HIV-1 dynamics reflect an intricate balance within the viruses’ host. The virus relies on host replication factors, but must escape or counter its host’s antiviral restriction factors. The interaction between the HIV-1 protein Vif and many cellular restriction factors from the APOBEC3 protein family is a prominent example of this evolutionary arms race. The viral infectivity factor (Vif protein largely neutralizes APOBEC3 proteins, which can induce in vivo hypermutations in HIV-1 to the extent of lethal mutagenesis, and ensures the production of viable virus particles. HIV-1 also uses the APOBEC3-Vif interaction to modulate its own mutation rate in harsh or variable environments, and it is a model of adaptation in a coevolutionary setting. Both experimental evidence and the substantiation of the underlying dynamics through coevolutionary models are presented as complementary views of a coevolutionary arms race.

  9. First Streptococcus pyogenes signature-tagged mutagenesis screen identifies novel virulence determinants.

    Science.gov (United States)

    Kizy, Anne E; Neely, Melody N

    2009-05-01

    The virulence of bacterial pathogens is a complex process that requires the dynamic expression of many genes for the pathogens to invade and circumvent host defenses, as well as to proliferate in vivo. In this study, we employed a large-scale screen, signature-tagged mutagenesis (STM), to identify Streptococcus pyogenes virulence genes important for pathogenesis within the host. Approximately 1,200 STM mutants were created and screened using the zebrafish infectious disease model. The transposon insertion site was identified for 29 of the 150 mutants that were considered attenuated for virulence. Previously reported streptococcal virulence genes, such as mga, hasA, amrA, smeZ, and two genes in the sil locus, were identified, confirming the utility of the model for revealing genes important for virulence. Multiple genes not previously implicated in virulence were also identified, including genes encoding putative transporters, hypothetical cytosolic proteins, and macrolide efflux pumps. The STM mutant strains display various levels of attenuation, and multiple separate insertions were identified in either the same gene or the same locus, suggesting that these factors are important for this type of acute, invasive infection. We further examined two such genes, silB and silC of a putative quorum-sensing regulon, and determined that they are significant virulence factors in our model of necrotizing fasciitis. sil locus promoter expression was examined under various in vitro conditions, as well as in zebrafish tissues, and was found to be differentially induced. This study was a unique investigation of S. pyogenes factors required for successful invasive infection.

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

    Directory of Open Access Journals (Sweden)

    Accardi Luisa

    2007-01-01

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

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

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    Gaynor Miller

    Full Text Available 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, 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. 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

  12. Site-directed Mutagenesis Improves the Thermostability and Specific-activity of Phytase Mutated by F43Y and I354M, L358F%F43Y及I354M,L358F定点突变对植酸酶热稳定性及酶活性的改善

    Institute of Scientific and Technical Information of China (English)

    陈惠; 王红宁; 杨婉身; 赵海霞; 吴琦; 单志

    2005-01-01

    对重组酵母PP-NPm-8的植酸酶phyAm基因进行PCR介导的定点突变,即将植酸酶43位的苯丙氨酸替换为酪氨酸(F43Y),将其354、358位的异亮氨酸、亮氨酸分别替换为甲硫氨酸和苯丙氨酸(I354M,L358F),得到了2个突变体PP-NPm-1(F43Y)及PP-NPm-2(I354M,L358F).含突变基因的重组表达载体pPIC 9k-phyAm-1,pPIC 9k-phyAm-2在毕赤酵母GS115中表达,对表达产物进行酶活性测定及热稳定性检测.结果表明:突变体PP-NPm-1最适反应温度比未突变体PP-NPm-8上升了3℃,75℃处理10 min,热稳定性提高15%,比活力提高11%;PP-NPm-2最适反应温度未改变,热稳定性比PP-NPm-8仅提高3%,比活力降低6.5%.对突变前后的植酸酶空间结构进行比较预测,发现突变氨基酸Tyr43与空间位置相邻的Asn416之间形成氢键,增强了酶的热稳定性.

  13. 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.

  14. The Site-directed Mutagenesis and Construction of A Highly Productive Expression Vector of the Cecropin Gene, An Antimicrobial Peptide From the Housefly (Musca domestica)%家蝇抗菌肽天蚕素基因的定点突变和高效表达质粒的构建

    Institute of Scientific and Technical Information of China (English)

    许小霞; 徐兴耀; 金丰良; 张古忍; 张文庆

    2004-01-01

    运用反转录聚合酶链式反应(RT-PCR)技术从家蝇体内扩增出抗菌肽天蚕素(cecropin)基因的开放阅读框(ORF),与pMD-18T载体重组,经限制性酶切片段分析和核苷酸序列分析,与GenBank中报道的序列一致.根据此ORF,重新合成1对引物,并在碳末端进行定点突变,加上Asn编码,使其末端酰氨化,再利用半嵌套式PCR扩增出家蝇cecropin基因的成熟肽,与双酶切的酵母表达载体pPICZαA连接,经PCR和双酶切鉴定,成功构建了分泌型表达质粒.

  15. 盐生盐杆菌RM07 DNA片段在大肠杆菌中的定点诱变和启动子功能分析%Site-directed Mutagenesis and Promoter Functional Analysis of RM07 DNA Fragment from Halobacterium halobium in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    杨洋; 沈萍

    2004-01-01

    将来源于嗜盐古生菌--盐生盐杆菌(Halobacterium halobium)基因组的RM07 DNA片段以正反两个方向分别插人大肠杆菌启动子探针载体pKK232-8携带的报告基因--氯霉素抗性基因(cat)的上游,得到RM07-cat融合的质粒pRM07-1(+)和pRM07-1(-),将其分别转入大肠杆菌HB101,进而检测了不同转化子菌株的氯霉素抗性水平和细胞内氯霉素乙酰转移酶蛋白质浓度.结果表明:正向的RM07片段在真细菌(大肠杆菌)中具有启动子活性,能够驱动cat报告基因的表达;而反向的RM07片段在大肠杆菌中不具有启动子活性.对RM07片段进行了定点诱变分析,检测了特定核苷酸突变对启动子活性的影响,结果进一步精确定位了RM07片段中对在大肠杆菌中的启动子功能有重要作用的关键碱基,并且通过改造RM07片段的碱基组成成分大幅提高了其在大肠杆菌中的启动子活性.

  16. Strategies used for genetically modifying bacterial genome:site-directed mutagenesis, gene inactivation, and gene over-expression%题目:遗传改造细菌基因组的策略:基因定点突变、基因失活和基因过表达

    Institute of Scientific and Technical Information of China (English)

    Jian-zhong XU; Wei-guo ZHANG‡

    2016-01-01

    概该综述较为全面地概述了当前针对大肠杆菌和谷氨酸棒杆菌基因组遗传改造的各个方法的具体流程、应用范围、注意事项以及其新颖之处,比较了针对基因定点突变、基因失活和基因过表达的各个方法所存在的优缺点,同时简单地介绍了利用质粒介导基因过表达所存在的问题。此外,还介绍了四种引物设计软件,并简单分析了它们的应用范围。为拟计划开展分子生物学实验的新手对关于细菌基因组遗传改造方法做了可靠的介绍,同时也为已进行相关实验的实验员提供关于基因定点突变、基因失活和基因过表达的最新信息。%With the availability of the whole genome sequence of Escherichia coli orCorynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in un-derstanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, alow a wide variety of DNA manipu-lation. However, the over-expression of the desired gene is generaly executed via plasmid-mediation. The current review summarizes the common strategies used for geneticaly modifyingE. coli andC. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via inte-grating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.

  17. Site-directed Mutagenesis of Putative Adenylylation Site of Glutamine Synthetase and Glutamine Production%谷氨酰胺合成酶腺苷酰化位点的定点突变和产胺的初步研究

    Institute of Scientific and Technical Information of China (English)

    黄星; 曾行; 刘铭; 龚博; 胡淼; 曹竹安

    2008-01-01

    为解决谷氨酰胺合成酶腺苷酰化修饰失活的问题,利用基因定点突变的方法将谷氨酸棒杆菌的谷氨酰胺合成酶(Glutamine Synthetase,GS)腺苷酰化位点由Tyr405突变为Phe405,并在大肠杆菌中获得突变后GS的表达-对比腺苷酰化位点突变前后的重组大肠杆菌pET-3a/GSI和pET-3a/GSIM在高氨环境下的GS活性和谷氨酰胺产量,发现重组菌pET-3a/GSIM在高氨环境下的最大酶活是150 U/L,产谷氨酰胺浓度为17.5 g/L,分别是pET-3a/GSI酶活(30 U/L)的5-0倍和产谷氨酰胺水平(3-4 g/L)的5-1倍,GS定点突变使谷氨酸转化为谷氨酰胺的途径得到强化-

  18. D190V点突变提高华根霉Rhizopus chinensis CCTCC M201021脂肪酶的最适温度和热稳定性%Improved optimum temperature and thermostability of the lipase from Rhizopus chinensis CCTCC M201021 by site-directed mutagenesis of D190V

    Institute of Scientific and Technical Information of China (English)

    吴厚军; 喻晓蔚; 沙冲; 徐岩

    2013-01-01

    [目的]对来源于Rhizopus chinensis CCTCC M201021的脂肪酶进行了D190V定点突变,提高该酶的最适温度和热稳定性.[方法]对毕赤酵母表达的突变酶D190V与野生型酶r27RCL进行酶学性质比较.[结果]D190V的最适温度比r27RCL高5℃,65℃下的半衰期提高了一倍,在其他性质方面,突变酶D190V与r27RCL基本相似.[结论]通过结构分析表明,定点突变D190V提高该酶稳定性的主要原因可能在于提高了突变位点所在的α螺旋的稳定性以及增强了稳定蛋白质结构的氢键作用力.

  19. Use of signature-tagged mutagenesis to identify virulence determinants in Haemophilus ducreyi responsible for ulcer formation.

    Science.gov (United States)

    Yeung, Angela; Cameron, D William; Desjardins, Marc; Lee, B Craig

    2011-02-01

    Elucidating the molecular mechanisms responsible for chancroid, a genital ulcer disease caused by Haemophilus ducreyi, has been hampered in part by the relative genetic intractability of the organism. A whole genome screen using signature-tagged mutagenesis in the temperature-dependent rabbit model (TDRM) of H. ducreyi infection uncovered 26 mutants with a presumptive attenuated phenotype. Insertions in two previously recognized virulence determinants, hgbA and lspA1, validated this genome scanning technique. Database interrogation allowed assignment of 24 mutants to several functional classes, including transport, metabolism, DNA repair, stress response and gene regulation. The attenuated virulence for a 3 strain with a mutation in hicB was confirmed by individual infection in the TDRM. The results from this preliminary study indicate that this high throughput strategy will further the understanding of the pathogenesis of H. ducreyi infection.

  20. 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.…

  1. 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.

  2. A mutagenesis screen in the mouse: Identification of novel gene functions in embryonic development

    NARCIS (Netherlands)

    Wansleeben, C.

    2010-01-01

    Developmental genetics has been a field of interest for over a hundred years. We have set out to perform an ENU mutagenesis screen in the mouse at E10.5. In the first chapter the pathways and developmental processes described in this thesis are introduced. In the second chapter we describe the ENU-m

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

    NARCIS (Netherlands)

    Wansleeben, C.; van Gurp, L.; Feitsma, H.; Kroon, C.; Rieter, E.; Verberne, M.; Guryev, V.; Cuppen, E.; Meijlink, F.

    2011-01-01

    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 we

  4. 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

    2007-01-01

    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 t

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

    Directory of Open Access Journals (Sweden)

    Francesca Valetti

    2013-10-01

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2006-09-01

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

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

    NARCIS (Netherlands)