Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

PubMed

Katano, Yuta; Li, Tongyang; Baba, Misato; Nakamura, Miyo; Ito, Masaaki; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

2017-12-01

We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

Dissecting partner recognition by an intrinsically disordered protein using descriptive random mutagenesis.

PubMed

Gruet, Antoine; Dosnon, Marion; Vassena, Andrea; Lombard, Vincent; Gerlier, Denis; Bignon, Christophe; Longhi, Sonia

2013-09-23

In view of getting insights into the molecular determinants of the binding efficiency of intrinsically disordered proteins (IDPs), we used random mutagenesis. As a proof of concept, we chose the interaction between the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) and the X domain (XD) of the viral phosphoprotein and assessed how amino acid substitutions introduced at random within NTAIL affect partner recognition. In contrast with directed evolution approaches, we did not apply any selection and used the gene library approach not for production purposes but for achieving a better understanding of the NTAIL/XD interaction. For that reason, and to differentiate our approach from similar approaches that make use of systematic (i.e., targeted) mutagenesis, we propose to call it "descriptive random mutagenesis" (DRM). NTAIL variants generated by error-prone PCR were picked at random in the absence of selection pressure and were characterized in terms of sequence and binding abilities toward XD. DRM not only identified determinants of NTAIL/XD interaction that were in good agreement with previous work but also provided new insights. In particular, we discovered that the primary interaction site is poorly evolvable in terms of binding abilities toward XD. We also identified a critical NTAIL residue whose role in stabilizing the NTAIL/XD complex had previously escaped detection, and we identified NTAIL regulatory sites that dampen the interaction while being located outside the primary interaction site. Results show that DRM is a valuable approach to study binding abilities of IDPs. © 2013 Elsevier Ltd. All rights reserved.

Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation mutagenesis.

PubMed

Kille, Sabrina; Acevedo-Rocha, Carlos G; Parra, Loreto P; Zhang, Zhi-Gang; Opperman, Diederik J; Reetz, Manfred T; Acevedo, Juan Pablo

2013-02-15

Saturation mutagenesis probes define sections of the vast protein sequence space. However, even if randomization is limited this way, the combinatorial numbers problem is severe. Because diversity is created at the codon level, codon redundancy is a crucial factor determining the necessary effort for library screening. Additionally, due to the probabilistic nature of the sampling process, oversampling is required to ensure library completeness as well as a high probability to encounter all unique variants. Our trick employs a special mixture of three primers, creating a degeneracy of 22 unique codons coding for the 20 canonical amino acids. Therefore, codon redundancy and subsequent screening effort is significantly reduced, and a balanced distribution of codon per amino acid is achieved, as demonstrated exemplarily for a library of cyclohexanone monooxygenase. We show that this strategy is suitable for any saturation mutagenesis methodology to generate less-redundant libraries.

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

PubMed

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.

[Stress-induced cellular adaptive mutagenesis].

PubMed

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.

Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis

PubMed Central

Valetti, Francesca; Gilardi, Gianfranco

2013-01-01

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

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

PubMed

Mrázková, Jana; Malinovská, Lenka; Wimmerová, Michaela

2017-01-01

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

Combinatorial multispectral, thermodynamics, docking and site-directed mutagenesis reveal the cognitive characteristics of honey bee chemosensory protein to plant semiochemical.

PubMed

Tan, Jing; Song, Xinmi; Fu, Xiaobin; Wu, Fan; Hu, Fuliang; Li, Hongliang

2018-08-05

In the chemoreceptive system of insects, there are always some soluble binding proteins, such as some antennal-specific chemosensory proteins (CSPs), which are abundantly distributed in the chemosensory sensillar lymph. The antennal-specific CSPs usually have strong capability to bind diverse semiochemicals, while the detailed interaction between CSPs and the semiochemicals remain unclear. Here, by means of the combinatorial multispectral, thermodynamics, docking and site-directed mutagenesis, we detailedly interpreted a binding interaction between a plant semiochemical β-ionone and antennal-specific CSP1 from the worker honey bee. Thermodynamic parameters (ΔH < 0, ΔS > 0) indicate that the interaction is mainly driven by hydrophobic forces and electrostatic interactions. Docking prediction results showed that there are two key amino acids, Phe44 and Gln63, may be involved in the interacting process of CSP1 to β-ionone. In order to confirm the two key amino acids, site-directed mutagenesis were performed and the binding constant (K A ) for two CSP1 mutant proteins was reduced by 60.82% and 46.80% compared to wild-type CSP1. The thermodynamic analysis of mutant proteins furtherly verified that Phe44 maintained an electrostatic interaction and Gln63 contributes hydrophobic and electrostatic forces. Our investigation initially elucidates the physicochemical mechanism of the interaction between antennal-special CSPs in insects including bees to plant semiochemicals, as well as the development of twice thermodynamic analysis (wild type and mutant proteins) combined with multispectral and site-directed mutagenesis methods. Copyright © 2018 Elsevier B.V. All rights reserved.

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

PubMed Central

Gurskaya, Nadya G; Savitsky, Alexander P; Yanushevich, Yurii G; Lukyanov, Sergey A; Lukyanov, Konstantin A

2001-01-01

Background Green Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (~ 485 nm), green (~ 505 nm), yellow (~ 540 nm), and red (>580 nm) emitters. Results Here we applied site-directed mutagenesis in order to investigate the structural background of color variety and possibility of shifting between different types of fluorescence. First, a blue-shifted mutant of cyan amFP486 was generated. Second, it was established that cyan and green emitters can be modified so as to produce an intermediate spectrum of fluorescence. Third, the relationship between green and yellow fluorescence was inspected on closely homologous green zFP506 and yellow zFP538 proteins. The following transitions of colors were performed: yellow to green; yellow to dual color (green and yellow); and green to yellow. Fourth, we generated a mutant of cyan emitter dsFP483 that demonstrated dual color (cyan and red) fluorescence. Conclusions Several amino acid substitutions were found to strongly affect fluorescence maxima. Some positions primarily found by sequence comparison were proved to be crucial for fluorescence of particular color. These results are the first step towards predicting the color of natural GFP-like proteins corresponding to newly identified cDNAs from corals. PMID:11459517

Multiple pathways for SOS-induced mutagenesis in Escherichia coli: An overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA

PubMed Central

Kim, Su-Ryang; Maenhaut-Michel, Geneviéve; Yamada, Masami; Yamamoto, Yoshihiro; Matsui, Keiko; Sofuni, Toshio; Nohmi, Takehiko; Ohmori, Haruo

1997-01-01

dinP is an Escherichia coli gene recently identified at 5.5 min of the genetic map, whose product shows a similarity in amino acid sequence to the E. coli UmuC protein involved in DNA damage-induced mutagenesis. In this paper we show that the gene is identical to dinB, an SOS gene previously localized near the lac locus at 8 min, the function of which was shown to be required for mutagenesis of nonirradiated λ phage infecting UV-preirradiated bacterial cells (termed λUTM for λ untargeted mutagenesis). A newly constructed dinP null mutant exhibited the same defect for λUTM as observed previously with a dinB::Mu mutant, and the defect was complemented by plasmids carrying dinP as the only intact bacterial gene. Furthermore, merely increasing the dinP gene expression, without UV irradiation or any other DNA-damaging treatment, resulted in a strong enhancement of mutagenesis in F′lac plasmids; at most, 800-fold increase in the G6-to-G5 change. The enhanced mutagenesis did not depend on recA, uvrA, or umuDC. Thus, our results establish that E. coli has at least two distinct pathways for SOS-induced mutagenesis: one dependent on umuDC and the other on dinB/P. PMID:9391106

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pletneva, Nadya V.; Pletnev, Vladimir Z., E-mail: vzpletnev@gmail.com; Souslova, Ekaterina

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 adoptsmore » 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.« less

PCR-mediated site-directed mutagenesis.

PubMed

Carey, Michael F; Peterson, Craig L; Smale, Stephen T

2013-08-01

Unlike traditional site-directed mutagenesis, this protocol requires only a single PCR step using full plasmid amplification to generate point mutants. The method can introduce small mutations into promoter sites and is even better suited for introducing single or double mutations into proteins. It is elegant in its simplicity and can be applied quite easily in any laboratory using standard protein expression vectors and commercially available reagents.

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

PubMed Central

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

2016-01-01

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

The Bacteriophage P1 HumD Protein Is a Functional Homolog of the Prokaryotic UmuD′-Like Proteins and Facilitates SOS Mutagenesis in Escherichia coli

PubMed Central

McLenigan, Mary P.; Kulaeva, Olga I.; Ennis, Don G.; Levine, Arthur S.; Woodgate, Roger

1999-01-01

The Escherichia coli umuD and umuC genes comprise an operon and encode proteins that are involved in the mutagenic bypass of normally replication-inhibiting DNA lesions. UmuD is, however, unable to function in this process until it undergoes a RecA-mediated cleavage reaction to generate UmuD′. Many homologs of umuDC have now been identified. Most are located on bacterial chromosomes or on broad-host-range R plasmids. One such putative homolog, humD (homolog of umuD) is, however, found on the bacteriophage P1 genome. Interestingly, humD differs from other umuD homologs in that it encodes a protein similar in size to the posttranslationally generated UmuD′ protein and not UmuD, nor is it in an operon with a cognate umuC partner. To determine if HumD is, in fact, a bona fide homolog of the prokaryotic UmuD′-like mutagenesis proteins, we have analyzed the ability of HumD to complement UmuD′ functions in vivo as well as examined HumD’s physical properties in vitro. When expressed from a high-copy-number plasmid, HumD restored cellular mutagenesis and increased UV survival to normally nonmutable recA430 lexA(Def) and UV-sensitive ΔumuDC recA718 lexA(Def) strains, respectively. Complementing activity was reduced when HumD was expressed from a low-copy-number plasmid, but this observation is explained by immunoanalysis which indicates that HumD is normally poorly expressed in vivo. In vitro analysis revealed that like UmuD′, HumD forms a stable dimer in solution and is able to interact with E. coli UmuC and RecA nucleoprotein filaments. We conclude, therefore, that bacteriophage P1 HumD is a functional homolog of the UmuD′-like proteins, and we speculate as to the reasons why P1 might require the activity of such a protein in vivo. PMID:10559166

Economical analysis of saturation mutagenesis experiments

PubMed Central

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

2015-01-01

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

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

PubMed Central

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

2014-01-01

Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although non-complementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts. PMID:24615866

Structure-based design of combinatorial mutagenesis libraries.

PubMed

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

2015-05-01

The development of protein variants with improved properties (thermostability, binding affinity, catalytic activity, etc.) has greatly benefited from the application of high-throughput screens evaluating large, diverse combinatorial libraries. At the same time, since only a very limited portion of sequence space can be experimentally constructed and tested, an attractive possibility is to use computational protein design to focus libraries on a productive portion of the space. We present a general-purpose method, called "Structure-based Optimization of Combinatorial Mutagenesis" (SOCoM), which can optimize arbitrarily large combinatorial mutagenesis libraries directly based on structural energies of their constituents. SOCoM chooses both positions and substitutions, employing a combinatorial optimization framework based on library-averaged energy potentials in order to avoid explicitly modeling every variant in every possible library. In case study applications to green fluorescent protein, β-lactamase, and lipase A, SOCoM optimizes relatively small, focused libraries whose variants achieve energies comparable to or better than previous library design efforts, as well as larger libraries (previously not designable by structure-based methods) whose variants cover greater diversity while still maintaining substantially better energies than would be achieved by representative random library approaches. By allowing the creation of large-scale combinatorial libraries based on structural calculations, SOCoM promises to increase the scope of applicability of computational protein design and improve the hit rate of discovering beneficial variants. While designs presented here focus on variant stability (predicted by total energy), SOCoM can readily incorporate other structure-based assessments, such as the energy gap between alternative conformational or bound states. © 2015 The Protein Society.

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

PubMed Central

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

2002-01-01

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

Genetic selection for a highly functional cysteine-less membrane protein using site-saturation mutagenesis

PubMed Central

Arendt, Cassandra S.; Ri, Keirei; Yates, Phillip A.; Ullman, Buddy

2007-01-01

We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site-saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2, in order to facilitate biochemical studies using thiol-specific modifying reagents. Of ten endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site-saturation mutagenesis scheme based on the Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in S. cerevisiae cells auxotrophic for purines, several highly functional non-cysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site-saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position. PMID:17481563

[Cloning, mutagenesis and symbiotic phenotype of three lipid transfer protein encoding genes from Mesorhizobium huakuii 7653R].

PubMed

Li, Yanan; Zeng, Xiaobo; Zhou, Xuejuan; Li, Youguo

2016-12-04

Lipid transfer protein superfamily is involved in lipid transport and metabolism. This study aimed to construct mutants of three lipid transfer protein encoding genes in Mesorhizobium huakuii 7653R, and to study the phenotypes and function of mutations during symbiosis with Astragalus sinicus. We used bioinformatics to predict structure characteristics and biological functions of lipid transfer proteins, and conducted semi-quantitative and fluorescent quantitative real-time PCR to analyze the expression levels of target genes in free-living and symbiotic conditions. Using pK19mob insertion mutagenesis to construct mutants, we carried out pot plant experiments to observe symbiotic phenotypes. MCHK-5577, MCHK-2172 and MCHK-2779 genes encoding proteins belonged to START/RHO alpha_C/PITP/Bet_v1/CoxG/CalC (SRPBCC) superfamily, involved in lipid transport or metabolism, and were identical to M. loti at 95% level. Gene relative transcription level of the three genes all increased compared to free-living condition. We obtained three mutants. Compared with wild-type 7653R, above-ground biomass of plants and nodulenitrogenase activity induced by the three mutants significantly decreased. Results indicated that lipid transfer protein encoding genes of Mesorhizobium huakuii 7653R may play important roles in symbiotic nitrogen fixation, and the mutations significantly affected the symbiotic phenotypes. The present work provided a basis to study further symbiotic function mechanism associated with lipid transfer proteins from rhizobia.

Quantitative Missense Variant Effect Prediction Using Large-Scale Mutagenesis Data.

PubMed

Gray, Vanessa E; Hause, Ronald J; Luebeck, Jens; Shendure, Jay; Fowler, Douglas M

2018-01-24

Large datasets describing the quantitative effects of mutations on protein function are becoming increasingly available. Here, we leverage these datasets to develop Envision, which predicts the magnitude of a missense variant's molecular effect. Envision combines 21,026 variant effect measurements from nine large-scale experimental mutagenesis datasets, a hitherto untapped training resource, with a supervised, stochastic gradient boosting learning algorithm. Envision outperforms other missense variant effect predictors both on large-scale mutagenesis data and on an independent test dataset comprising 2,312 TP53 variants whose effects were measured using a low-throughput approach. This dataset was never used for hyperparameter tuning or model training and thus serves as an independent validation set. Envision prediction accuracy is also more consistent across amino acids than other predictors. Finally, we demonstrate that Envision's performance improves as more large-scale mutagenesis data are incorporated. We precompute Envision predictions for every possible single amino acid variant in human, mouse, frog, zebrafish, fruit fly, worm, and yeast proteomes (https://envision.gs.washington.edu/). Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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

2016-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a powerful tool for genome editing in numerous organisms. However, the system is typically used for gene editing throughout the entire organism. Tissue and temporal specific mutagenesis is often desirable to determine gene function in a specific stage or tissue and to bypass undesired consequences of global mutations. We have developed the CRISPR/Cas system for conditional mutagenesis in transgenic zebrafish using tissue-specific and/or inducible expression of Cas9 and U6-driven expression of sgRNA. To allow mutagenesis of multiple targets, we have isolated four distinct U6 promoters and designed Golden Gate vectors to easily assemble transgenes with multiple sgRNAs. We provide experimental details on the reagents and applications for multiplex conditional mutagenesis in zebrafish. Copyright © 2016 Elsevier Inc. All rights reserved.

Automated use of mutagenesis data in structure prediction.

PubMed

Nanda, Vikas; DeGrado, William F

2005-05-15

In the absence of experimental structural determination, numerous methods are available to indirectly predict or probe the structure of a target molecule. Genetic modification of a protein sequence is a powerful tool for identifying key residues involved in binding reactions or protein stability. Mutagenesis data is usually incorporated into the modeling process either through manual inspection of model compatibility with empirical data, or through the generation of geometric constraints linking sensitive residues to a binding interface. We present an approach derived from statistical studies of lattice models for introducing mutation information directly into the fitness score. The approach takes into account the phenotype of mutation (neutral or disruptive) and calculates the energy for a given structure over an ensemble of sequences. The structure prediction procedure searches for the optimal conformation where neutral sequences either have no impact or improve stability and disruptive sequences reduce stability relative to wild type. We examine three types of sequence ensembles: information from saturation mutagenesis, scanning mutagenesis, and homologous proteins. Incorporating multiple sequences into a statistical ensemble serves to energetically separate the native state and misfolded structures. As a result, the prediction of structure with a poor force field is sufficiently enhanced by mutational information to improve accuracy. Furthermore, by separating misfolded conformations from the target score, the ensemble energy serves to speed up conformational search algorithms such as Monte Carlo-based methods. Copyright 2005 Wiley-Liss, Inc.

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

PubMed

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

2016-01-01

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

Significantly improving the yield of recombinant proteins in Bacillus subtilis by a novel powerful mutagenesis tool (ARTP): Alkaline α-amylase as a case study.

PubMed

Ma, Yingfang; Yang, Haiquan; Chen, Xianzhong; Sun, Bo; Du, Guocheng; Zhou, Zhemin; Song, Jiangning; Fan, You; Shen, Wei

2015-10-01

In this study, atmospheric and room temperature plasma (ARTP), a promising mutation breeding technique, was successfully applied to generate Bacillus subtilis mutants that yielded large quantities of recombinant protein. The high throughput screening platform was implemented to select those mutants with the highest yield of recombinant alkaline α-amylase (AMY), including the preferred mutant B. subtilis WB600 mut-12#. The yield and productivity of recombinant AMY in B. subtilis WB600 mut-12# increased 35.0% and 8.8%, respectively, the extracellular protein concentration of which increased 37.9%. B. subtilis WB600 mut-12# exhibited good genetic stability. Cells from B. subtilis WB600 mut-12# became shorter and wider than those from the wild-type. This study is the first to report a novel powerful mutagenesis tool (ARTP) that significantly improves the yield of recombinant proteins in B. subtilis and may therefore play an important role in the high expression level of proteins in recombinant microbial hosts. Copyright © 2015 Elsevier Inc. All rights reserved.

Mutant fatty acid desaturase and methods for directed mutagenesis

DOEpatents

Shanklin, John [Shoreham, NY; Whittle, Edward J [Greenport, NY

2008-01-29

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

Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line.

PubMed

Feitsma, Harma; de Bruijn, Ewart; van de Belt, Jose; Nijman, Isaac J; Cuppen, Edwin

2008-07-01

S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.

Environmental stress induces trinucleotide repeat mutagenesis in human cells.

PubMed

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

2015-03-24

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

Biophysical Optimization of a Therapeutic Protein by Nonstandard Mutagenesis

PubMed Central

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

2014-01-01

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

Modelling of microbial polyhydroxyalkanoate surface binding protein PhaP for rational mutagenesis.

PubMed

Zhao, Hongyu; Yao, Zhenyu; Chen, Xiangbin; Wang, Xinquan; Chen, Guo-Qiang

2017-11-01

Phasins are unusual amphiphilic proteins that bind to microbial polyhydroxyalkanoate (PHA) granules in nature and show great potential for various applications in biotechnology and medicine. Despite their remarkable diversity, only the crystal structure of PhaP A h from Aeromonas hydrophila has been solved to date. Based on the structure of PhaP A h , homology models of PhaP A z from Azotobacter sp. FA-8 and PhaP TD from Halomonas bluephagenesis TD were successfully established, allowing rational mutagenesis to be conducted to enhance the stability and surfactant properties of these proteins. PhaP A z mutants, including PhaP A z Q38L and PhaP A z Q78L, as well as PhaP TD mutants, including PhaP TD Q38M and PhaP TD Q72M, showed better emulsification properties and improved thermostability (6-10°C higher melting temperatures) compared with their wild-type homologues under the same conditions. Importantly, the established PhaP homology-modelling approach, based on the high-resolution structure of PhaP A h , can be generalized to facilitate the study of other PhaP members. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Development of a Dunaliella tertiolecta Strain with Increased Zeaxanthin Content Using Random Mutagenesis

PubMed Central

Kim, Minjae; Ahn, Junhak; Jeon, Hancheol; Jin, EonSeon

2017-01-01

Zeaxanthin is a xanthophyll pigment that is regarded as one of the best carotenoids for the prevention and treatment of degenerative diseases. In the worldwide natural products market, consumers prefer pigments that have been produced from biological sources. In this study, a Dunaliella tertiolecta strain that has 10–15% higher cellular zeaxanthin content than the parent strain (zea1), was obtained by random mutagenesis using ethyl methanesulfonate (EMS) as a mutagen. This mutant, mp3, was grown under various salinities and light intensities to optimize culture conditions for zeaxanthin production. The highest cellular zeaxanthin content was observed at 1.5 M NaCl and 65–85 μmol photons·m−2·s−1, and the highest daily zeaxanthin productivity was observed at 0.6 M NaCl and 140–160 μmol photons·m−2·s−1. The maximal yield of zeaxanthin from mp3 in fed-batch culture was 8 mg·L−1, which was obtained at 0.6 M NaCl and 140–160 μmol photons·m−2·s−1. These results suggest that random mutagenesis with EMS is useful for generating D. tertiolecta strains with increased zeaxanthin content, and also suggest optimal culture conditions for the enhancement of biomass and zeaxanthin production by the zeaxanthin accumulating mutant strains. PMID:28635662

Development of a Dunaliella tertiolecta Strain with Increased Zeaxanthin Content Using Random Mutagenesis.

PubMed

Kim, Minjae; Ahn, Junhak; Jeon, Hancheol; Jin, EonSeon

2017-06-21

Zeaxanthin is a xanthophyll pigment that is regarded as one of the best carotenoids for the prevention and treatment of degenerative diseases. In the worldwide natural products market, consumers prefer pigments that have been produced from biological sources. In this study, a Dunaliella tertiolecta strain that has 10-15% higher cellular zeaxanthin content than the parent strain ( zea1 ), was obtained by random mutagenesis using ethyl methanesulfonate (EMS) as a mutagen. This mutant, mp3 , was grown under various salinities and light intensities to optimize culture conditions for zeaxanthin production. The highest cellular zeaxanthin content was observed at 1.5 M NaCl and 65-85 μmol photons·m -2 ·s -1 , and the highest daily zeaxanthin productivity was observed at 0.6 M NaCl and 140-160 μmol photons·m -2 ·s -1 . The maximal yield of zeaxanthin from mp3 in fed-batch culture was 8 mg·L -1 , which was obtained at 0.6 M NaCl and 140-160 μmol photons·m -2 ·s -1 . These results suggest that random mutagenesis with EMS is useful for generating D. tertiolecta strains with increased zeaxanthin content, and also suggest optimal culture conditions for the enhancement of biomass and zeaxanthin production by the zeaxanthin accumulating mutant strains.

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

PubMed

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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hawwa, Renda; Larsen, Sonia D.; Ratia, Kiira

2010-11-09

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

Novel Escherichia coli umuD′ Mutants: Structure-Function Insights into SOS Mutagenesis

PubMed Central

McLenigan, Mary; Peat, Thomas S.; Frank, Ekaterina G.; McDonald, John P.; Gonzalez, Martín; Levine, Arthur S.; Hendrickson, Wayne A.; Woodgate, Roger

1998-01-01

Although it has been 10 years since the discovery that the Escherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD′, the function of UmuD′ has yet to be determined. In an attempt to elucidate the role of UmuD′ in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD′ plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD′ mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD′, three were nonsense mutations that resulted in a truncated UmuD′ protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal umuD1, umuD44, and umuD77 alleles into umuD′. All 17 umuD′ mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD′. PMID:9721309

Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR.

PubMed

Dumas, Louis; Zito, Francesca; Auroy, Pascaline; Johnson, Xenie; Peltier, Gilles; Alric, Jean

2018-06-01

Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b 6 f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast. © 2018 American Society of Plant Biologists. All rights reserved.

Environmental stress induces trinucleotide repeat mutagenesis in human cells

PubMed Central

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

2015-01-01

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

Novel Random Mutagenesis Method for Directed Evolution.

PubMed

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.

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

PubMed

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

2017-01-01

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

Back to the future: revisiting HIV-1 lethal mutagenesis

PubMed Central

Dapp, Michael J.; Patterson, Steven E.; Mansky, Louis M.

2012-01-01

The concept of eliminating HIV-1 infectivity by elevating the viral mutation rate was first proposed over a decade ago, even though the general concept had been conceived earlier for RNA viruses. Lethal mutagenesis was originally viewed as a novel chemotherapeutic approach for treating HIV-1 infection in which use of a viral mutagen would over multiple rounds of replication lead to the lethal accumulation of mutations, rendering the virus population non infectious – known as the slow mutation accumulation model. There have been limitations in obtaining good efficacy data with drug leads, leaving some doubt into clinical translation. More recent studies of the APOBEC3 proteins as well as new progress in the use of nucleoside analogs for inducing lethal mutagenesis have helped to refocus attention on rapid induction of HIV-1 lethal mutagenesis in a single or limited number of replication cycles leading to a rapid mutation accumulation model. PMID:23195922

Favipiravir elicits antiviral mutagenesis during virus replication in vivo.

PubMed

Arias, Armando; Thorne, Lucy; Goodfellow, Ian

2014-10-21

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

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

PubMed

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

2011-10-01

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

Facile Site-Directed Mutagenesis of Large Constructs Using Gibson Isothermal DNA Assembly.

PubMed

Yonemoto, Isaac T; Weyman, Philip D

2017-01-01

Site-directed mutagenesis is a commonly used molecular biology technique to manipulate biological sequences, and is especially useful for studying sequence determinants of enzyme function or designing proteins with improved activity. We describe a strategy using Gibson Isothermal DNA Assembly to perform site-directed mutagenesis on large (>~20 kbp) constructs that are outside the effective range of standard techniques such as QuikChange II (Agilent Technologies), but more reliable than traditional cloning using restriction enzymes and ligation.

Concomitant Lethal Mutagenesis of Human Immunodeficiency Virus Type 1

PubMed Central

Dapp, Michael J.; Holtz, Colleen M.; Mansky, Louis M.

2012-01-01

RNA virus population dynamics is complex, and sophisticated approaches are needed in many cases for therapeutic intervention. One such approach, termed lethal mutagenesis, is directed at targeting the virus population structure for extinction or error catastrophe. Previous studies have demonstrated the concept of this approach with human immunodeficiency virus type 1 (HIV-1) by use of chemical mutagens (i.e., 5-azacytidine) as well as by host factors with mutagenic properties (i.e., APOBEC3G). In this study, these two unrelated mutagenic agents were used concomitantly to investigate the interplay of these distinct mutagenic mechanisms. Specifically, an HIV-1 was produced from APOBEC3G (A3G)-expressing cells and used to infect permissive target cells treated with 5-azacytidine (5-AZC). Reduced viral infectivity and increased viral mutagenesis was observed with both the viral mutagen (i.e., G-to-C mutations) and the host restriction factor (i.e., G-to-A mutations); however, when combined, had complex interactions. Intriguingly, nucleotide sequence analysis revealed that concomitant HIV-1 exposure to both 5-AZC and A3G resulted in an increase of G-to-A viral mutagenesis at the expense of G-to-C mutagenesis. A3G catalytic activity was required for the diminution in G-to-C mutagenesis. Taken together, our findings provide the first demonstration for potentiation of the mutagenic effect of a cytosine analog by A3G expression, resulting in concomitant HIV-1 lethal mutagenesis. PMID:22426127

Chemical and UV Mutagenesis.

PubMed

Bose, Jeffrey L

2016-01-01

The ability to create mutations is an important step towards understanding bacterial physiology and virulence. While targeted approaches are invaluable, the ability to produce genome-wide random mutations can lead to crucial discoveries. Transposon mutagenesis is a useful approach, but many interesting mutations can be missed by these insertions that interrupt coding and noncoding sequences due to the integration of an entire transposon. Chemical mutagenesis and UV-based random mutagenesis are alternate approaches to isolate mutations of interest with the potential of only single nucleotide changes. Once a standard method, difficulty in identifying mutation sites had decreased the popularity of this technique. However, thanks to the recent emergence of economical whole-genome sequencing, this approach to making mutations can once again become a viable option. Therefore, this chapter provides an overview protocol for random mutagenesis using UV light or DNA-damaging chemicals.

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

PubMed

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

2016-01-01

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

Site-directed mutagenesis of the hinge peptide from the hemagglutinin protein: enhancement of the pH-responsive conformational change.

PubMed

Casali, Monica; Banta, Scott; Zambonelli, Carlo; Megeed, Zaki; Yarmush, Martin L

2008-06-01

Environmentally responsive proteins and peptides are increasingly finding utility in various engineered systems due to their ability to respond to the presentation of external stimuli. A classic example of this behavior is the influenza hemagglutinin (HA) fusion protein. At neutral pH, HA exists in a non-fusogenic state, but upon exposure to low pH, the conformation of the structure changes to expose a fusogenic peptide. During this structural change, massive rearrangements occur in a subunit of HA (HA2). Crystallography data has shown that a loop of 28 amino acids (residues 54-81) undergoes a dramatic transition from a random coil to an alpha-helix. This segment connects to two flanking helical regions (short and long) to form a long, continuous helix. Here, we report the results of site-directed mutagenesis study on LOOP-36 to further understand the mechanism of this important stimulus-responsive peptide. The conformational transition of a bacterially expressed LOOP-36 was found to be less dramatic than has been previously reported. The systematic mutation of glutamate and histidine residues in the peptide to glutamines (glutamine scanning) did not impact the conformational behavior of the peptide, but the substitution of the glycine residue at position 22 with alanine resulted in significant pH-responsive behavior. Therefore this mutant stimulus-responsive peptide may be more valuable for future protein engineering and bionanotechnology efforts.

Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

PubMed

Taniguchi, Naohiro; Murakami, Hiroshi

2017-01-01

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

Facile Affinity Maturation of Antibody Variable Domains Using Natural Diversity Mutagenesis

PubMed Central

Tiller, Kathryn E.; Chowdhury, Ratul; Li, Tong; Ludwig, Seth D.; Sen, Sabyasachi; Maranas, Costas D.; Tessier, Peter M.

2017-01-01

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

Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

PubMed Central

Dror, Adi; Shemesh, Einav; Dayan, Natali

2014-01-01

The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production. PMID:24362426

Construction of a mutagenesis cartridge for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuhn, R.J.; Tada, H.; Ypma-Wong, M.F.

1988-01-01

By following a strategy of genetic analysis of poliovirus, the authors have constructed a synthetic mutagenesis cartridge spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type I (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed them to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alterationmore » of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. They suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).« less

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

PubMed

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

2012-03-01

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

Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly.

PubMed

Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen; Jespersen, Jakob Berg; Sommer, Morten O A; Wernersson, Rasmus; Olsen, Lars Rønn

2015-03-20

USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at http://www.cbs.dtu.dk/services/AMUSER/.

Lysine-Based Site-Directed Mutagenesis Increased Rigid β-Sheet Structure and Thermostability of Mesophilic 1,3-1,4-β-Glucanase.

PubMed

Niu, Chengtuo; Zhu, Linjiang; Zhu, Pei; Li, Qi

2015-06-03

1,3-1,4-β-Glucanase is widely applied in the food industry, while its low thermostability often reduces its performance. In a previous study, chemical modification of surface lysine residues was proved to increase the thermostability of β-glucanase. To improve the thermostability, the mesophilic β-glucanase from Bacillus terquilensis was rationally engineered through site-directed mutagenesis of the 12 lysines into serines. The results showed that the K20S, K117S, and K165S mutants could both enhance the specific activities and thermostability of β-glucanase. The triple mutant (K20S/K117S/K165S) could increase the optimal temperature and T50 value by 15 and 14 °C, respectively. Five percent more structured residues were observed in the mutant, which formed new β-sheet structures in the concave side. Molecular dynamics simulation analysis showed that the flexibility in the mutation regions was decreased, which resulted in the overall rigidity of the β-glucanase. Therefore, the lysine-based site-directed mutagenesis is a simple and effective method for improving the thermostability of β-glucanase.

Towards the construction of high-quality mutagenesis libraries.

PubMed

Li, Heng; Li, Jing; Jin, Ruinan; Chen, Wei; Liang, Chaoning; Wu, Jieyuan; Jin, Jian-Ming; Tang, Shuang-Yan

2018-07-01

To improve the quality of mutagenesis libraries in directed evolution strategy. In the process of library transformation, transformants which have been shown to take up more than one plasmid might constitute more than 20% of the constructed library, thereby extensively impairing the quality of the library. We propose a practical transformation method to prevent the occurrence of multiple-plasmid transformants while maintaining high transformation efficiency. A visual library model containing plasmids expressing different fluorescent proteins was used. Multiple-plasmid transformants can be reduced through optimizing plasmid DNA amount used for transformation based on the positive correlation between the occurrence frequency of multiple-plasmid transformants and the logarithmic ratio of plasmid molecules to competent cells. This method provides a simple solution for a seemingly common but often neglected problem, and should be valuable for improving the quality of mutagenesis libraries to enhance the efficiency of directed evolution strategies.

Forward and reverse mutagenesis in C. elegans

PubMed Central

Kutscher, Lena M.; Shaham, Shai

2014-01-01

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

Saturation mutagenesis reveals manifold determinants of exon definition.

PubMed

Ke, Shengdong; Anquetil, Vincent; Zamalloa, Jorge Rojas; Maity, Alisha; Yang, Anthony; Arias, Mauricio A; Kalachikov, Sergey; Russo, James J; Ju, Jingyue; Chasin, Lawrence A

2018-01-01

To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem-loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins. © 2018 Ke et al.; Published by Cold Spring Harbor Laboratory Press.

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

PubMed

Lee, Raymond Teck Ho; Ng, Ashley Shu Mei; Ingham, Philip W

2016-01-01

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

Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases.

PubMed

Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

2015-10-16

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.

Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases

PubMed Central

Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

2015-01-01

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

Transcriptional mutagenesis: causes and involvement in tumor development

PubMed Central

Brégeon, Damien; Doetsch, Paul W.

2013-01-01

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

Mutagenesis Studies of the H5 Influenza Hemagglutinin Stem Loop Region*

PubMed Central

Antanasijevic, Aleksandar; Basu, Arnab; Bowlin, Terry L.; Mishra, Rama K.; Rong, Lijun; Caffrey, Michael

2014-01-01

Influenza outbreaks, particularly the pandemic 1918 H1 and avian H5 strains, are of high concern to public health. The hemagglutinin envelope protein of influenza plays a critical role in viral entry and thus is an attractive target for inhibition of virus entry. The highly conserved stem loop region of hemagglutinin has been shown to undergo critically important conformational changes during the entry process and, moreover, to be a site for inhibition of virus entry by antibodies, small proteins, and small drug-like molecules. In this work we probe the structure-function properties of the H5 hemagglutinin stem loop region by site-directed mutagenesis. We find that most mutations do not disrupt expression, proteolytic processing, incorporation into virus, or receptor binding; however, many of the mutations disrupt the entry process. We further assess the effects of mutations on inhibition of entry by a neutralizing monoclonal antibody (C179) and find examples of increased and decreased sensitivity to the antibody, consistent with the antibody binding site observed by x-ray crystallography. In addition, we tested the sensitivity of the mutants to MBX2329, a small molecule inhibitor of influenza entry. Interestingly, the mutants exhibit increased and decreased sensitivities to MBX2329, which gives further insight into the binding site of the compound on HA and potential mechanisms of escape. Finally, we have modeled the binding site of MBX2329 using molecular dynamics and find that the resulting structure is in good agreement with the mutagenesis results. Together these studies underscore the importance of the stem loop region to HA function and suggest potential sites for therapeutic intervention of influenza entry. PMID:24947513

Optogenetic mutagenesis in Caenorhabditis elegans.

PubMed

Noma, Kentaro; Jin, Yishi

2015-12-03

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.

Optogenetic mutagenesis in Caenorhabditis elegans

PubMed Central

Noma, Kentaro; Jin, Yishi

2015-01-01

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

Effective lethal mutagenesis of influenza virus by three nucleoside analogs.

PubMed

Pauly, Matthew D; Lauring, Adam S

2015-04-01

Lethal mutagenesis is a broad-spectrum antiviral strategy that exploits the high mutation rate and low mutational tolerance of many RNA viruses. This approach uses mutagenic drugs to increase viral mutation rates and burden viral populations with mutations that reduce the number of infectious progeny. We investigated the effectiveness of lethal mutagenesis as a strategy against influenza virus using three nucleoside analogs, ribavirin, 5-azacytidine, and 5-fluorouracil. All three drugs were active against a panel of seasonal H3N2 and laboratory-adapted H1N1 strains. We found that each drug increased the frequency of mutations in influenza virus populations and decreased the virus' specific infectivity, indicating a mutagenic mode of action. We were able to drive viral populations to extinction by passaging influenza virus in the presence of each drug, indicating that complete lethal mutagenesis of influenza virus populations can be achieved when a sufficient mutational burden is applied. Population-wide resistance to these mutagenic agents did not arise after serial passage of influenza virus populations in sublethal concentrations of drug. Sequencing of these drug-passaged viral populations revealed genome-wide accumulation of mutations at low frequency. The replicative capacity of drug-passaged populations was reduced at higher multiplicities of infection, suggesting the presence of defective interfering particles and a possible barrier to the evolution of resistance. Together, our data suggest that lethal mutagenesis may be a particularly effective therapeutic approach with a high genetic barrier to resistance for influenza virus. Influenza virus is an RNA virus that causes significant morbidity and mortality during annual epidemics. Novel therapies for RNA viruses are needed due to the ease with which these viruses evolve resistance to existing therapeutics. Lethal mutagenesis is a broad-spectrum strategy that exploits the high mutation rate and the low

Mutagenesis: Interactions with a parallel universe.

PubMed

Miller, Jeffrey H

Unexpected observations in mutagenesis research have led to a new perspective in this personal reflection based on years of studying mutagenesis. Many mutagens have been thought to operate via a single principal mechanism, with secondary effects usually resulting in only minor changes in the observed mutation frequencies and spectra. For example, we conceive of base analogs as resulting in direct mispairing as their main mechanism of mutagenesis. Recent studies now show that in fact even these simple mutagens can cause very large and unanticipated effects both in mutation frequencies and in the mutational spectra when used in certain pair-wise combinations. Here we characterize this leap in mutation frequencies as a transport to an alternate universe of mutagenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

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

PubMed

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

2014-01-01

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

New Parameters for Higher Accuracy in the Computation of Binding Free Energy Differences upon Alanine Scanning Mutagenesis on Protein-Protein Interfaces.

PubMed

Simões, Inês C M; Costa, Inês P D; Coimbra, João T S; Ramos, Maria J; Fernandes, Pedro A

2017-01-23

Knowing how proteins make stable complexes enables the development of inhibitors to preclude protein-protein (P:P) binding. The identification of the specific interfacial residues that mostly contribute to protein binding, denominated as hot spots, is thus critical. Here, we refine an in silico alanine scanning mutagenesis protocol, based on a residue-dependent dielectric constant version of the Molecular Mechanics/Poisson-Boltzmann Surface Area method. We have used a large data set of structurally diverse P:P complexes to redefine the residue-dependent dielectric constants used in the determination of binding free energies. The accuracy of the method was validated through comparison with experimental data, considering the per-residue P:P binding free energy (ΔΔG binding ) differences upon alanine mutation. Different protocols were tested, i.e., a geometry optimization protocol and three molecular dynamics (MD) protocols: (1) one using explicit water molecules, (2) another with an implicit solvation model, and (3) a third where we have carried out an accelerated MD with explicit water molecules. Using a set of protein dielectric constants (within the range from 1 to 20) we showed that the dielectric constants of 7 for nonpolar and polar residues and 11 for charged residues (and histidine) provide optimal ΔΔG binding predictions. An overall mean unsigned error (MUE) of 1.4 kcal mol -1 relative to the experiment was achieved in 210 mutations only with geometry optimization, which was further reduced with MD simulations (MUE of 1.1 kcal mol -1 for the MD employing explicit solvent). This recalibrated method allows for a better computational identification of hot spots, avoiding expensive and time-consuming experiments or thermodynamic integration/ free energy perturbation/ uBAR calculations, and will hopefully help new drug discovery campaigns in their quest of searching spots of interest for binding small drug-like molecules at P:P interfaces.

Transcription coupled repair deficiency protects against human mutagenesis and carcinogenesis: Personal Reflections on the 50th anniversary of the discovery of xeroderma pigmentosum.

PubMed

Cleaver, James E

2017-10-01

Xeroderma pigmentosum (XP) patients who lack the main damage recognition protein for global genome repair (GGR), XPC, have greatly increased skin cancer rates and elevated mutation frequencies originating from unrepaired ultraviolet photoproducts in the nontranscribed regions of the genome and in nontranscribed strands of expressed genes. But they show no increased mutations in transcribed strands. In contrast, cancer is absent from Cockayne syndrome (CS) patients that have defective transcription coupled repair (TCR) despite severe photosensitivity, CS patients remarkably show no elevation of UV induced mutagenesis implying that defective TCR may be protective against mutagenesis and carcinogenesis. Mutation avoidance in CS is postulated to occur through arrested transcription that generates a tripled stranded R loop consisting of DNA double strands and a nascent mRNA strand. R loops result in S phase apoptosis or activation of ATM kinase that causes a delay in DNA replication until TCR, or transcript cleavage by TFIIS or RNAaseH, relieves the transcription block. Resumption of replication then occurs on repaired DNA without concomitant mutagenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2017-01-01

Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, E. coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. PMID:27234563

Coupling unbiased mutagenesis to high-throughput DNA sequencing uncovers functional domains in the Ndc80 kinetochore protein of Saccharomyces cerevisiae.

PubMed

Tien, Jerry F; Fong, Kimberly K; Umbreit, Neil T; Payen, Celia; Zelter, Alex; Asbury, Charles L; Dunham, Maitreya J; Davis, Trisha N

2013-09-01

During mitosis, kinetochores physically link chromosomes to the dynamic ends of spindle microtubules. This linkage depends on the Ndc80 complex, a conserved and essential microtubule-binding component of the kinetochore. As a member of the complex, the Ndc80 protein forms microtubule attachments through a calponin homology domain. Ndc80 is also required for recruiting other components to the kinetochore and responding to mitotic regulatory signals. While the calponin homology domain has been the focus of biochemical and structural characterization, the function of the remainder of Ndc80 is poorly understood. Here, we utilized a new approach that couples high-throughput sequencing to a saturating linker-scanning mutagenesis screen in Saccharomyces cerevisiae. We identified domains in previously uncharacterized regions of Ndc80 that are essential for its function in vivo. We show that a helical hairpin adjacent to the calponin homology domain influences microtubule binding by the complex. Furthermore, a mutation in this hairpin abolishes the ability of the Dam1 complex to strengthen microtubule attachments made by the Ndc80 complex. Finally, we defined a C-terminal segment of Ndc80 required for tetramerization of the Ndc80 complex in vivo. This unbiased mutagenesis approach can be generally applied to genes in S. cerevisiae to identify functional properties and domains.

The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

PubMed Central

Kozmin, Stanislav G.; Jinks-Robertson, Sue

2013-01-01

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. PMID:23307894

Mutagenesis studies of the H5 influenza hemagglutinin stem loop region.

PubMed

Antanasijevic, Aleksandar; Basu, Arnab; Bowlin, Terry L; Mishra, Rama K; Rong, Lijun; Caffrey, Michael

2014-08-08

Influenza outbreaks, particularly the pandemic 1918 H1 and avian H5 strains, are of high concern to public health. The hemagglutinin envelope protein of influenza plays a critical role in viral entry and thus is an attractive target for inhibition of virus entry. The highly conserved stem loop region of hemagglutinin has been shown to undergo critically important conformational changes during the entry process and, moreover, to be a site for inhibition of virus entry by antibodies, small proteins, and small drug-like molecules. In this work we probe the structure-function properties of the H5 hemagglutinin stem loop region by site-directed mutagenesis. We find that most mutations do not disrupt expression, proteolytic processing, incorporation into virus, or receptor binding; however, many of the mutations disrupt the entry process. We further assess the effects of mutations on inhibition of entry by a neutralizing monoclonal antibody (C179) and find examples of increased and decreased sensitivity to the antibody, consistent with the antibody binding site observed by x-ray crystallography. In addition, we tested the sensitivity of the mutants to MBX2329, a small molecule inhibitor of influenza entry. Interestingly, the mutants exhibit increased and decreased sensitivities to MBX2329, which gives further insight into the binding site of the compound on HA and potential mechanisms of escape. Finally, we have modeled the binding site of MBX2329 using molecular dynamics and find that the resulting structure is in good agreement with the mutagenesis results. Together these studies underscore the importance of the stem loop region to HA function and suggest potential sites for therapeutic intervention of influenza entry. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Mutagenesis of Trichoderma Viride by Ultraviolet and Plasma

NASA Astrophysics Data System (ADS)

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

2012-04-01

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

Himar1 Transposon for Efficient Random Mutagenesis in Aggregatibacter actinomycetemcomitans

PubMed Central

Ding, Qinfeng; Tan, Kai Soo

2017-01-01

Aggregatibacter actinomycetemcomitans is the primary etiological agent of aggressive periodontal disease. Identification of novel virulence factors at the genome-wide level is hindered by lack of efficient genetic tools to perform mutagenesis in this organism. The Himar1 mariner transposon is known to yield a random distribution of insertions in an organism’s genome with requirement for only a TA dinucleotide target and is independent of host-specific factors. However, the utility of this system in A. actinomycetemcomitans is unknown. In this study, we found that Himar1 transposon mutagenesis occurs at a high frequency (×10-4), and can be universally applied to wild-type A. actinomycetemcomitans strains of serotypes a, b, and c. The Himar1 transposon inserts were stably inherited in A. actinomycetemcomitans transconjugants in the absence of antibiotics. A library of 16,000 mutant colonies of A. actinomycetemcomitans was screened for reduced biofilm formation. Mutants with transposon inserts in genes encoding pilus, putative ion transporters, multidrug resistant proteins, transcription regulators and enzymes involved in the synthesis of extracellular polymeric substance, bacterial metabolism and stress response were discovered in this screen. Our results demonstrated the utility of the Himar1 mutagenesis system as a novel genetic tool for functional genomic analysis in A. actinomycetemcomitans. PMID:29018421

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

PubMed Central

Ilmjärv, Tanel; Naanuri, Eve; Kivisaar, Maia

2017-01-01

Bacteria can rapidly evolve mechanisms allowing them to use toxic environmental pollutants as a carbon source. In the current study we examined whether the survival and evolution of indigenous bacteria with the capacity to degrade organic pollutants could be connected with increased mutation frequency. The presence of constitutive and transient mutators was monitored among 53 pollutants-degrading indigenous bacterial strains. Only two strains expressed a moderate mutator phenotype and six were hypomutators, which implies that constitutively increased mutability has not been prevalent in the evolution of pollutants degrading bacteria. At the same time, a large proportion of the studied indigenous strains exhibited UV-irradiation-induced mutagenesis, indicating that these strains possess error-prone DNA polymerases which could elevate mutation frequency transiently under the conditions of DNA damage. A closer inspection of two Pseudomonas fluorescens strains PC20 and PC24 revealed that they harbour genes for ImuC (DnaE2) and more than one copy of genes for Pol V. Our results also revealed that availability of other nutrients in addition to aromatic pollutants in the growth environment of bacteria affects mutagenic effects of aromatic compounds. These results also implied that mutagenicity might be affected by a factor of how long bacteria have evolved to use a particular pollutant as a carbon source. PMID:28777807

Structure-based design of combinatorial mutagenesis libraries

PubMed Central

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

2015-01-01

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

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

PubMed

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

2016-01-01

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

High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor expression and function.

PubMed

Bill, Anke; Rosethorne, Elizabeth M; Kent, Toby C; Fawcett, Lindsay; Burchell, Lynn; van Diepen, Michiel T; Marelli, Anthony; Batalov, Sergey; Miraglia, Loren; Orth, Anthony P; Renaud, Nicole A; Charlton, Steven J; Gosling, Martin; Gaither, L Alex; Groot-Kormelink, Paul J

2014-01-01

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function

PubMed Central

Kent, Toby C.; Fawcett, Lindsay; Burchell, Lynn; van Diepen, Michiel T.; Marelli, Anthony; Batalov, Sergey; Miraglia, Loren; Orth, Anthony P.; Renaud, Nicole A.; Charlton, Steven J.; Gosling, Martin; Gaither, L. Alex; Groot-Kormelink, Paul J.

2014-01-01

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs. PMID:24886841

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

PubMed

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

2012-09-01

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

DinB Upregulation Is the Sole Role of the SOS Response in Stress-Induced Mutagenesis in Escherichia coli

PubMed Central

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

2009-01-01

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

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

PubMed Central

Varshney, Gaurav Kumar

2014-01-01

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

Effect of SOS-induced levels of imuABC on spontaneous and damage-induced mutagenesis in Caulobacter crescentus.

PubMed

Alves, Ingrid R; Lima-Noronha, Marco A; Silva, Larissa G; Fernández-Silva, Frank S; Freitas, Aline Luiza D; Marques, Marilis V; Galhardo, Rodrigo S

2017-11-01

imuABC (imuAB dnaE2) genes are responsible for SOS-mutagenesis in Caulobacter crescentus and other bacterial species devoid of umuDC. In this work, we have constructed operator-constitutive mutants of the imuABC operon. We used this genetic tool to investigate the effect of SOS-induced levels of these genes upon both spontaneous and damage-induced mutagenesis. We showed that constitutive expression of imuABC does not increase spontaneous or damage-induced mutagenesis, nor increases cellular resistance to DNA-damaging agents. Nevertheless, the presence of the operator-constitutive mutation rescues mutagenesis in a recA background, indicating that imuABC are the only genes required at SOS-induced levels for translesion synthesis (TLS) in C. crescentus. Furthermore, these data also show that TLS mediated by ImuABC does not require RecA, unlike umuDC-dependent mutagenesis in E. coli. Copyright © 2017 Elsevier B.V. All rights reserved.

Lethal mutagenesis: targeting the mutator phenotype in cancer.

PubMed

Fox, Edward J; Loeb, Lawrence A

2010-10-01

The evolution of cancer and RNA viruses share many similarities. Both exploit high levels of genotypic diversity to enable extensive phenotypic plasticity and thereby facilitate rapid adaptation. In order to accumulate large numbers of mutations, we have proposed that cancers express a mutator phenotype. Similar to cancer cells, many viral populations, by replicating their genomes with low fidelity, carry a substantial mutational load. As high levels of mutation are potentially deleterious, the viral mutation frequency is thresholded at a level below which viral populations equilibrate in a traditional mutation-selection balance, and above which the population is no longer viable, i.e., the population undergoes an error catastrophe. Because their mutation frequencies are fine-tuned just below this error threshold, viral populations are susceptible to further increases in mutational load and, recently this phenomenon has been exploited therapeutically by a concept that has been termed lethal mutagenesis. Here we review the application of lethal mutagenesis to the treatment of HIV and discuss how lethal mutagenesis may represent a novel therapeutic approach for the treatment of solid cancers. Copyright © 2010 Elsevier Ltd. All rights reserved.

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

USDA-ARS?s Scientific Manuscript database

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

Visualization of tandem repeat mutagenesis in Bacillus subtilis.

PubMed

Dormeyer, Miriam; Lentes, Sabine; Ballin, Patrick; Wilkens, Markus; Klumpp, Stefan; Kohlheyer, Dietrich; Stannek, Lorena; Grünberger, Alexander; Commichau, Fabian M

2018-03-01

Mutations are crucial for the emergence and evolution of proteins with novel functions, and thus for the diversity of life. Tandem repeats (TRs) are mutational hot spots that are present in the genomes of all organisms. Understanding the molecular mechanism underlying TR mutagenesis at the level of single cells requires the development of mutation reporter systems. Here, we present a mutation reporter system that is suitable to visualize mutagenesis of TRs occurring in single cells of the Gram-positive model bacterium Bacillus subtilis using microfluidic single-cell cultivation. The system allows measuring the elimination of TR units due to growth rate recovery. The cultivation of bacteria carrying the mutation reporter system in microfluidic chambers allowed us for the first time to visualize the emergence of a specific mutation at the level of single cells. The application of the mutation reporter system in combination with microfluidics might be helpful to elucidate the molecular mechanism underlying TR (in)stability in bacteria. Moreover, the mutation reporter system might be useful to assess whether mutations occur in response to nutrient starvation. Copyright © 2018 Elsevier B.V. All rights reserved.

Genome editing using CRISPR/Cas9-targeted mutagenesis: An opportunity for yield improvements of crop plants grown under environmental stresses.

PubMed

Abdelrahman, Mostafa; Al-Sadi, Abdullah M; Pour-Aboughadareh, Alireza; Burritt, David J; Tran, Lam-Son Phan

2018-03-12

Developing more crops able to sustainably produce high yields when grown under biotic/abiotic stresses is an important goal, if crop production and food security are to be guaranteed in the face of ever-increasing human population and unpredictable global climatic conditions. However, conventional crop improvement, through random mutagenesis or genetic recombination, is time-consuming and cannot keep pace with increasing food demands. Targeted genome editing (GE) technologies, especially clustered regularly interspaced short palindromic repeats (CRISPR)/(CRISPR)-associated protein 9 (Cas9), have great potential to aid in the breeding of crops that are able to produce high yields under conditions of biotic/abiotic stress. This is due to their high efficiency, accuracy and low risk of off-target effects, compared with conventional random mutagenesis methods. The use of CRISPR/Cas9 system has grown very rapidly in recent years with numerous examples of targeted mutagenesis in crop plants, including gene knockouts, modifications, and the activation and repression of target genes. The potential of the GE approach for crop improvement has been clearly demonstrated. However, the regulation and social acceptance of GE crops still remain a challenge. In this review, we evaluate the recent applications of the CRISPR/Cas9-mediated GE, as a means to produce crop plants with greater resilience to the stressors they encounter when grown under increasing stressful environmental conditions. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

PubMed

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

2016-06-01

CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo. © 2016. Published by The Company of Biologists Ltd.

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

PubMed

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

2009-11-01

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

Tnt1 Retrotransposon Mutagenesis: A Tool for Soybean Functional Genomics1[W][OA

PubMed Central

Cui, Yaya; Barampuram, Shyam; Stacey, Minviluz G.; Hancock, C. Nathan; Findley, Seth; Mathieu, Melanie; Zhang, Zhanyuan; Parrott, Wayne A.; Stacey, Gary

2013-01-01

Insertional mutagenesis is a powerful tool for determining gene function in both model and crop plant species. Tnt1, the transposable element of tobacco (Nicotiana tabacum) cell type 1, is a retrotransposon that replicates via an RNA copy that is reverse transcribed and integrated elsewhere in the plant genome. Based on studies in a variety of plants, Tnt1 appears to be inactive in normal plant tissue but can be reactivated by tissue culture. Our goal was to evaluate the utility of the Tnt1 retrotransposon as a mutagenesis strategy in soybean (Glycine max). Experiments showed that the Tnt1 element was stably transformed into soybean plants by Agrobacterium tumefaciens-mediated transformation. Twenty-seven independent transgenic lines carrying Tnt1 insertions were generated. Southern-blot analysis revealed that the copy number of transposed Tnt1 elements ranged from four to 19 insertions, with an average of approximately eight copies per line. These insertions showed Mendelian segregation and did not transpose under normal growth conditions. Analysis of 99 Tnt1 flanking sequences revealed insertions into 62 (62%) annotated genes, indicating that the element preferentially inserts into protein-coding regions. Tnt1 insertions were found in all 20 soybean chromosomes, indicating that Tnt1 transposed throughout the soybean genome. Furthermore, fluorescence in situ hybridization experiments validated that Tnt1 inserted into multiple chromosomes. Passage of transgenic lines through two different tissue culture treatments resulted in Tnt1 transposition, significantly increasing the number of insertions per line. Thus, our data demonstrate the Tnt1 retrotransposon to be a powerful system that can be used for effective large-scale insertional mutagenesis in soybean. PMID:23124322

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

PubMed

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

2018-02-01

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

CiPerGenesis, A Mutagenesis Approach that Produces Small Libraries of Circularly Permuted Proteins Randomly Opened at a Focused Region: Testing on the Green Fluorescent Protein.

PubMed

Gaytán, Paul; Roldán-Salgado, Abigail; Yáñez, Jorge A; Morales-Arrieta, Sandra; Juárez-González, Víctor R

2018-06-12

Circularly permuted proteins (cpPs) represent a novel type of mutant proteins with original termini that are covalently linked through a peptide connector and opened at any other place of the polypeptide backbone to create new ends. cpPs are finding wide applications in biotechnology because their properties may be quite different from those of the parental protein. However, the actual challenge for the creation of successful cpPs is to identify those peptide bonds that can be broken to create new termini and ensure functional and well-folded cpPs. Herein, we describe CiPerGenesis, a combinatorial mutagenesis approach that uses two oligonucleotide libraries to amplify a circularized gene by PCR, starting and ending from a focused target region. This approach creates small libraries of circularly permuted genes that are easily cloned in the correct direction and frame using two different restriction sites encoded in the oligonucleotides. Once expressed, the protein libraries exhibit a unique sequence diversity, comprising cpPs that exhibit ordinary breakpoints between adjacent amino acids localized at the target region as well as cpPs with new termini containing user-defined truncations and repeats of some amino acids. CiPerGenesis was tested at the lid region G134-H148 of green fluorescent protein (GFP), revealing that the most fluorescent variants were those starting at Leu141 and ending at amino acids Tyr145, Tyr143, Glu142, Leu141, Lys140, and H139. Purification and biochemical characterization of some variants suggested a differential expression, solubility and maturation extent of the mutant proteins as the likely cause for the variability in fluorescence intensity observed in colonies.

Recent advances of microbial breeding via heavy-ion mutagenesis at IMP.

PubMed

Hu, W; Li, W; Chen, J

2017-10-01

Nowadays, the value of heavy-ion mutagenesis has been accepted as a novel powerful mutagen technique to generate new microbial mutants due to its high linear energy transfer and high relative biological effectiveness. This paper briefly reviews recent progress in developing a more efficient mutagenesis technique for microbial breeding using heavy-ion mutagenesis, and also presents the outline of the beam line for microbial breeding in Heavy Ion Research Facility of Lanzhou. Then, new insights into microbial biotechnology via heavy-ion mutagenesis are also further explored. We hope that our concerns will give deep insight into microbial breeding biotechnology via heavy-ion mutagenesis. We also believe that heavy-ion mutagenesis breeding will greatly contribute to the progress of a comprehensive study industrial strain engineering for bioindustry in the future. There is currently a great interest in developing rapid and diverse microbial mutation tool for strain modification. Heavy-ion mutagenesis has been proved as a powerful technology for microbial breeding due to its broad spectrum of mutation phenotypes with high efficiency. In order to deeply understand heavy-ion mutagenesis technology, this paper briefly reviews recent progress in microbial breeding using heavy-ion mutagenesis at IMP, and also presents the outline of the beam line for microbial breeding in Heavy Ion Research Facility of Lanzhou (HIRFL) as well as new insights into microbial biotechnology via heavy-ion mutagenesis. Thus, this work can provide the guidelines to promote the development of novel microbial biotechnology cross-linking heavy-ion mutagenesis breeding that could make breeding process more efficiently in the future. © 2017 The Society for Applied Microbiology.

Elucidation of Lipid Binding Sites on Lung Surfactant Protein A Using X-ray Crystallography, Mutagenesis, and Molecular Dynamics Simulations.

PubMed

Goh, Boon Chong; Wu, Huixing; Rynkiewicz, Michael J; Schulten, Klaus; Seaton, Barbara A; McCormack, Francis X

2016-07-05

Surfactant protein A (SP-A) is a collagenous C-type lectin (collectin) that is critical for pulmonary defense against inhaled microorganisms. Bifunctional avidity of SP-A for pathogen-associated molecular patterns (PAMPs) such as lipid A and for dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant membranes lining the air-liquid interface of the lung, ensures that the protein is poised for first-line interactions with inhaled pathogens. To improve our understanding of the motifs that are required for interactions with microbes and surfactant structures, we explored the role of the tyrosine-rich binding surface on the carbohydrate recognition domain of SP-A in the interaction with DPPC and lipid A using crystallography, site-directed mutagenesis, and molecular dynamics simulations. Critical binding features for DPPC binding include a three-walled tyrosine cage that binds the choline headgroup through cation-π interactions and a positively charged cluster that binds the phosphoryl group. This basic cluster is also critical for binding of lipid A, a bacterial PAMP and target for SP-A. Molecular dynamics simulations further predict that SP-A binds lipid A more tightly than DPPC. These results suggest that the differential binding properties of SP-A favor transfer of the protein from surfactant DPPC to pathogen membranes containing appropriate lipid PAMPs to effect key host defense functions.

Orthogonal combinatorial mutagenesis: a codon-level combinatorial mutagenesis method useful for low multiplicity and amino acid-scanning protocols

PubMed Central

Gaytán, Paul; Yáñez, Jorge; Sánchez, Filiberto; Soberón, Xavier

2001-01-01

We describe here a method to generate combinatorial libraries of oligonucleotides mutated at the codon-level, with control of the mutagenesis rate so as to create predictable binomial distributions of mutants. The method allows enrichment of the libraries with single, double or larger multiplicity of amino acid replacements by appropriate choice of the mutagenesis rate, depending on the concentration of synthetic precursors. The method makes use of two sets of deoxynucleoside-phosphoramidites bearing orthogonal protecting groups [4,4′-dimethoxytrityl (DMT) and 9-fluorenylmethoxycarbonyl (Fmoc)] in the 5′ hydroxyl. These phosphoramidites are divergently combined during automated synthesis in such a way that wild-type codons are assembled with commercial DMT-deoxynucleoside-methyl-phosphoramidites while mutant codons are assembled with Fmoc-deoxynucleoside-methyl-phosphoramidites in an NNG/C fashion in a single synthesis column. This method is easily automated and suitable for low mutagenesis rates and large windows, such as those required for directed evolution and alanine scanning. Through the assembly of three oligonucleotide libraries at different mutagenesis rates, followed by cloning at the polylinker region of plasmid pUC18 and sequencing of 129 clones, we concluded that the method performs essentially as intended. PMID:11160911

Modification of Antibody Function by Mutagenesis.

PubMed

Dasch, James R; Dasch, Amy L

2017-09-01

The ability to "fine-tune" recombinant antibodies by mutagenesis separates recombinant antibodies from hybridoma-derived antibodies because the latter are locked with respect to their properties. Recombinant antibodies can be modified to suit the application: Changes in isotype, format (e.g., scFv, Fab, bispecific antibodies), and specificity can be made once the heavy- and light-chain sequences are available. After immunoglobulin heavy and light chains for a particular antibody have been cloned, the binding site-namely, the complementarity determining regions (CDR)-can be manipulated by mutagenesis to obtain antibody variants with improved properties. The method described here is relatively simple, uses commercially available reagents, and is effective. Using the pComb3H vector, a commercial mutagenesis kit, PfuTurbo polymerase (Agilent), and two mutagenic primers, a library of phage with mutagenized heavy and light CDR3 can be obtained. © 2017 Cold Spring Harbor Laboratory Press.

Posttranslational arginylation enzyme Ate1 affects DNA mutagenesis by regulating stress response

PubMed Central

Kumar, Akhilesh; Birnbaum, Michael D; Patel, Devang M; Morgan, William M; Singh, Jayanti; Barrientos, Antoni; Zhang, Fangliang

2016-01-01

Arginyltransferase 1 (Ate1) mediates protein arginylation, a poorly understood protein posttranslational modification (PTM) in eukaryotic cells. Previous evidence suggest a potential involvement of arginylation in stress response and this PTM was traditionally considered anti-apoptotic based on the studies of individual substrates. However, here we found that arginylation promotes cell death and/or growth arrest, depending on the nature and intensity of the stressing factor. Specifically, in yeast, mouse and human cells, deletion or downregulation of the ATE1 gene disrupts typical stress responses by bypassing growth arrest and suppressing cell death events in the presence of disease-related stressing factors, including oxidative, heat, and osmotic stresses, as well as the exposure to heavy metals or radiation. Conversely, in wild-type cells responding to stress, there is an increase of cellular Ate1 protein level and arginylation activity. Furthermore, the increase of Ate1 protein directly promotes cell death in a manner dependent on its arginylation activity. Finally, we found Ate1 to be required to suppress mutation frequency in yeast and mammalian cells during DNA-damaging conditions such as ultraviolet irradiation. Our study clarifies the role of Ate1/arginylation in stress response and provides a new mechanism to explain the link between Ate1 and a variety of diseases including cancer. This is also the first example that the modulation of the global level of a PTM is capable of affecting DNA mutagenesis. PMID:27685622

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

PubMed

Kashiwaba, Shu-ichiro; Kanao, Rie; Masuda, Yuji; Kusumoto-Matsuo, Rika; Hanaoka, Fumio; Masutani, Chikahide

2015-12-15

Mono-ubiquitinated PCNA activates error-prone DNA polymerases; therefore, strict regulation of PCNA mono-ubiquitination is crucial in avoiding undesired mutagenesis. In this study, we used an in vitro assay system to identify USP7 as a deubiquitinating enzyme of mono-ubiquitinated PCNA. Suppression of USP1, a previously identified PCNA deubiquitinase, or USP7 increased UV- and H2O2-induced PCNA mono-ubiquitination in a distinct and additive manner, suggesting that USP1 and USP7 make different contributions to PCNA deubiquitination in human cells. Cell-cycle-synchronization analyses revealed that USP7 suppression increased H2O2-induced PCNA ubiquitination throughout interphase, whereas USP1 suppression specifically increased ubiquitination in S-phase cells. UV-induced mutagenesis was elevated in USP1-suppressed cells, whereas H2O2-induced mutagenesis was elevated in USP7-suppressed cells. These results suggest that USP1 suppresses UV-induced mutations produced in a manner involving DNA replication, whereas USP7 suppresses H2O2-induced mutagenesis involving cell-cycle-independent processes such as DNA repair. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2016-04-18

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

Dynamic Fluctuations of Protein-Carbohydrate Interactions Promote Protein Aggregation

PubMed Central

Voynov, Vladimir; Chennamsetty, Naresh; Kayser, Veysel; Helk, Bernhard; Forrer, Kurt; Zhang, Heidi; Fritsch, Cornelius; Heine, Holger; Trout, Bernhardt L.

2009-01-01

Protein-carbohydrate interactions are important for glycoprotein structure and function. Antibodies of the IgG class, with increasing significance as therapeutics, are glycosylated at a conserved site in the constant Fc region. We hypothesized that disruption of protein-carbohydrate interactions in the glycosylated domain of antibodies leads to the exposure of aggregation-prone motifs. Aggregation is one of the main problems in protein-based therapeutics because of immunogenicity concerns and decreased efficacy. To explore the significance of intramolecular interactions between aromatic amino acids and carbohydrates in the IgG glycosylated domain, we utilized computer simulations, fluorescence analysis, and site-directed mutagenesis. We find that the surface exposure of one aromatic amino acid increases due to dynamic fluctuations. Moreover, protein-carbohydrate interactions decrease upon stress, while protein-protein and carbohydrate-carbohydrate interactions increase. Substitution of the carbohydrate-interacting aromatic amino acids with non-aromatic residues leads to a significantly lower stability than wild type, and to compromised binding to Fc receptors. Our results support a mechanism for antibody aggregation via decreased protein-carbohydrate interactions, leading to the exposure of aggregation-prone regions, and to aggregation. PMID:20037630

A practical teaching course in directed protein evolution using the green fluorescent protein as a model.

PubMed

Ruller, Roberto; Silva-Rocha, Rafael; Silva, Artur; Cruz Schneider, Maria Paula; Ward, Richard John

2011-01-01

Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from Aequorea victoria by a random mutagenesis strategy using error-prone polymerase chain reaction. Screening of bacterial colonies transformed with random mutant libraries identified GFP variants with increased fluorescence yields. Mapping the three-dimensional structure of these mutants demonstrated how alterations in structural features such as the environment around the fluorophore and properties of the protein surface can influence functional properties such as the intensity of fluorescence and protein solubility. Copyright © 2011 Wiley Periodicals, Inc.

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

PubMed

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

2015-11-01

Decitabine has previously been shown to induce lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1). However, the factors that determine the susceptibilities of individual sequence positions in HIV-1 to decitabine have not yet been defined. To investigate this, we performed Illumina high-throughput sequencing of multiple amplicons prepared from proviral DNA that was recovered from decitabine-treated cells infected with HIV-1. We found that decitabine induced an ≈4.1-fold increase in the total mutation frequency of HIV-1, primarily due to a striking ≈155-fold increase in the G-to-C transversion frequency. Intriguingly, decitabine also led to an ≈29-fold increase in the C-to-G transversion frequency. G-to-C frequencies varied substantially (up to ≈80-fold) depending upon sequence position, but surprisingly, mutational hot spots (defined as upper outliers within the mutation frequency distribution) were not observed. We further found that every single guanine position examined was significantly susceptible to the mutagenic effects of decitabine. Taken together, these observations demonstrate for the first time that decitabine-mediated HIV-1 mutagenesis is promiscuous and occurs in the absence of a clear bias for mutational hot spots. These data imply that decitabine-mediated G-to-C mutagenesis is a highly effective antiviral mechanism for extinguishing HIV-1 infectivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

PubMed

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.

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli

PubMed Central

Standley, Melissa; Allen, Jennifer; Cervantes, Layla; Lilly, Joshua; Camps, Manel

2017-01-01

Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. The present article focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inactivating mutation (reversion mutation assay). Both methods are labor-intensive, involving visual screening, quantification of colonies on solid media, or determining a Poisson distribution in liquid culture. Here we present two reversion reporters for in vivo mutagenesis that produce a quantitative output, and thus have the potential to greatly reduce the amount of test chemical and labor involved in these assays. This output is obtained by coupling a TEM β lactamase-based reversion assay with GFP fluorescence, either by placing the two genes on the same plasmid or by fusing them translationally and interrupting the N-terminus of the ORF with a stop codon. We also describe a reporter aimed at facilitating the monitoring of continuous mutagenesis in mutator strains. This reporter couples two reversion markers, allowing the temporal separation of mutation events in time, thus providing information about the dynamics of mutagenesis in mutator strains. Here, we describe these reporter systems, provide protocols for use, and demonstrate their key functional features using error-prone Pol I mutagenesis as a source of mutations. PMID:28645368

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli.

PubMed

Standley, Melissa; Allen, Jennifer; Cervantes, Layla; Lilly, Joshua; Camps, Manel

2017-01-01

Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. This chapter focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inactivating mutation (reversion mutation assay). Both methods are labor intensive, involving visual screening, quantification of colonies on solid media, or determining a Poisson distribution in liquid culture. Here, we present two reversion reporters for in vivo mutagenesis that produce a quantitative output, and thus have the potential to greatly reduce the amount of test chemical and labor involved in these assays. This output is obtained by coupling a TEM β lactamase-based reversion assay with GFP fluorescence, either by placing the two genes on the same plasmid or by fusing them translationally and interrupting the N-terminus of the chimeric ORF with a stop codon. We also describe a reporter aimed at facilitating the monitoring of continuous mutagenesis in mutator strains. This reporter couples two reversion markers, allowing the temporal separation of mutation events in time, thus providing information about the dynamics of mutagenesis in mutator strains. Here, we describe these reporter systems, provide protocols for use, and demonstrate their key functional features using error-prone Pol I mutagenesis as a source of mutations. © 2017 Elsevier Inc. All rights reserved.

Enhanced mutagenesis parallels enhanced reactivation of herpes virus in a human cell line.

PubMed Central

Lytle, C D; Knott, D C

1982-01-01

U.v. irradiation of human NB-E cells results in enhanced mutagenesis and enhanced reactivation of u.v.-irradiated H-1 virus grown in those cells ( Cornelis et al., 1982). This paper reports a similar study using herpes simplex virus (HSV) in NB-E cells. The mutation frequency of HSV (resistance of virus plaque formation to 40 micrograms/ml iododeoxycytidine ) increased approximately linearly with exposure of the virus to u.v. radiation. HSV grown in unirradiated cells gave a slope of 1.8 X 10(-5)m2/J, with 3.2 X 10(-5)m2/J for HSV grown in cells irradiated (3 J/m2) 24 h before infection. There was no evidence for mutagenesis of unirradiated virus by irradiated cells, as seen with H-1 virus. Enhanced reactivation of irradiated HSV in parallel cultures increased virus survival, manifested as a change in slope of the final component of the two-component survival curve from a D0 of 27 J/m2 in unirradiated cells to 45 J/m2 in irradiated cells. Thus, enhanced mutagenesis and enhanced reactivation occurred for irradiated HSV in NB-E cells. The difference in the enhanced mutagenesis of HSV (dependent on damaged DNA sites) and of H-1 virus (primarily independent of damaged DNA sites) is discussed in terms of differences in DNA polymerases. PMID:6329698

The Yeast Environmental Stress Response Regulates Mutagenesis Induced by Proteotoxic Stress

PubMed Central

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

2013-01-01

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

Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.

PubMed

Subburaj, Saminathan; Chung, Sung Jin; Lee, Choongil; Ryu, Seuk-Min; Kim, Duk Hyoung; Kim, Jin-Soo; Bae, Sangsu; Lee, Geung-Joo

2016-07-01

Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

PubMed Central

Huovinen, Tuomas; Brockmann, Eeva-Christine; Akter, Sultana; Perez-Gamarra, Susan; Ylä-Pelto, Jani; Liu, Yuan; Lamminmäki, Urpo

2012-01-01

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material. PMID:22355397

Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses

PubMed Central

Johnson, Stephen M.; Eltahla, Auda A.; Aloi, Maria; Aloia, Amanda L.; McDevitt, Christopher A.; Bull, Rowena A.

2017-01-01

ABSTRACT Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3′ untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies. IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In

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

PubMed

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

2016-08-01

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

Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content.

PubMed

Xu, Jingyu; Francis, Tammy; Mietkiewska, Elzbieta; Giblin, E Michael; Barton, Dennis L; Zhang, Yan; Zhang, Meng; Taylor, David C

2008-10-01

A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.

Mutagenesis of diploid mammalian genes by gene entrapment

PubMed Central

Lin, Qing; Donahue, Sarah L.; Moore-Jarrett, Tracy; Cao, Shang; Osipovich, Anna B.; Ruley, H. Earl

2006-01-01

The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector–cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice. Cells homozygous for most entrapment loci could be isolated by selecting for enhanced expression of an inserted neomycin-resistance gene that resulted from losses of heterozygosity (LOH). The frequencies of LOH measured at 37 sites in the genome ranged from 1.3 × 10−5 to 1.2 × 10−4 per cell and increased with increasing distance from the centromere, implicating mitotic recombination in the process. The ease and efficiency of obtaining homozygous mutations will (i) facilitate genetic studies of gene function in cultured cells, (ii) permit genome-wide studies of recombination events that result in LOH and mediate a type of chromosomal instability important in carcinogenesis, and (iii) provide new strategies for phenotype-driven mutagenesis screens in mammalian cells. PMID:17062627

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

PubMed

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

2015-09-25

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

Computer Simulation of Mutagenesis.

ERIC Educational Resources Information Center

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

1978-01-01

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

The contribution of Nth and Nei DNA glycosylases to mutagenesis in Mycobacterium smegmatis.

PubMed

Moolla, Nabiela; Goosens, Vivianne J; Kana, Bavesh D; Gordhan, Bhavna G

2014-01-01

The increased prevalence of drug resistant strains of Mycobacterium tuberculosis (Mtb) indicates that significant mutagenesis occurs during tuberculosis disease in humans. DNA damage by host-derived reactive oxygen/nitrogen species is hypothesized to be critical for the mutagenic process in Mtb thus, highlighting an important role for DNA repair enzymes in maintenance of genome fidelity. Formamidopyrimidine (Fpg/MutM/Fapy) and EndonucleaseVIII (Nei) constitute the Fpg/Nei family of DNA glycosylases and together with EndonucleaseIII (Nth) are central to the base excision repair pathway in bacteria. In this study we assess the contribution of Nei and Nth DNA repair enzymes in Mycobacterium smegmatis (Msm), which retains a single nth homologue and duplications of the Fpg (fpg1 and fpg2) and Nei (nei1 and nei2) homologues. Using an Escherichia coli nth deletion mutant, we confirm the functionality of the mycobacterial nth gene in the base excision repair pathway. Msm mutants lacking nei1, nei2 and nth individually or in combination did not display aberrant growth in broth culture. Deletion of nth individually results in increased UV-induced mutagenesis and combinatorial deletion with the nei homologues results in reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin. Deletion of nth together with the fpg homolgues did not result in any growth/survival defects or changes in mutation rate. Furthermore, no differential emergence of the common rifampicin resistance conferring genotypes were noted. Collectively, these data confirm a role for Nth in base excision repair in mycobacteria and further highlight a novel interplay between the Nth and Nei homologues in spontaneous mutagenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

Empirical complexities in the genetic foundations of lethal mutagenesis.

PubMed

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

2013-10-01

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

Molecular dynamics simulation studies and in vitro site directed mutagenesis of avian beta-defensin Apl_AvBD2

PubMed Central

2010-01-01

Background Defensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. To test this hypothesis and to improve the activity of the newly identified avian β-defensin Apl_AvBD2 by enhancing the cationicity, we performed in silico site directed mutagenesis, keeping the predicted secondary structure intact. Molecular dynamics (MD) simulation studies were done to predict the activity. Mutant proteins were made by in vitro site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis. Results MD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic in silico mutants, which were not detected in the initial structural prediction by homology modelling. The C-terminal cationic 'claw' region, important in antimicrobial activity, which was intact in the wild type, showed changes in shape and orientation in all the mutant peptides. Mutant peptides also showed increased solvent accessible surface area and more number of hydrogen bonds with the surrounding water molecules. In functional studies, the Escherichia coli expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein. Conclusion The study revealed that cationicity alone is not the determining factor in the microbicidal activity of antimicrobial peptides. Factors affecting the molecular dynamics such as hydrophobicity, electrostatic interactions and the potential for oligomerization may also play fundamental roles. It points to the usefulness of MD simulation studies in successful engineering of antimicrobial peptides for improved activity and other desirable functions. PMID

Dissecting protein function: an efficient protocol for identifying separation-of-function mutations that encode structurally stable proteins.

PubMed

Lubin, Johnathan W; Rao, Timsi; Mandell, Edward K; Wuttke, Deborah S; Lundblad, Victoria

2013-03-01

Mutations that confer the loss of a single biochemical property (separation-of-function mutations) can often uncover a previously unknown role for a protein in a particular biological process. However, most mutations are identified based on loss-of-function phenotypes, which cannot differentiate between separation-of-function alleles vs. mutations that encode unstable/unfolded proteins. An alternative approach is to use overexpression dominant-negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain when overexpressed. This is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are able to compete with the endogenous protein (Herskowitz 1987). To test this, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy, which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts. Reverse mutagenesis of EST3, which targeted different categories of amino acids, also showed that mutating highly conserved charged residues to the oppositely charged amino acid had an increased likelihood of generating a severely defective est3(-) mutation, which nevertheless encoded a structurally stable protein. These results suggest that charge-swap mutagenesis directed at a limited subset of highly conserved charged residues, combined with ODN screening to eliminate partially unfolded proteins, may provide a widely applicable and efficient strategy for generating separation-of-function mutations.

The Fanconi anemia pathway limits the severity of mutagenesis.

PubMed

Hinz, John M; Nham, Peter B; Salazar, Edmund P; Thompson, Larry H

2006-08-13

Fanconi anemia (FA) is a developmental and cancer predisposition disorder in which key, yet unknown, physiological events promoting chromosome stability are compromised. FA cells exhibit excess metaphase chromatid breaks and are universally hypersensitive to DNA interstrand crosslinking agents. Published mutagenesis data from single-gene mutation assays show both increased and decreased mutation frequencies in FA cells. In this review we discuss the data from the literature and from our isogenic fancg knockout hamster CHO cells, and interpret these data within the framework of a molecular model that accommodates these seemingly divergent observations. In FA cells, reduced rates of recovery of viable X-linked hypoxanthine phosphoribosyltransferase (hprt) mutants are characteristically observed for diverse mutagenic agents, but also in untreated cultures, indicating the relevance of the FA pathway for processing assorted DNA lesions. We ascribe these reductions to: (1) impaired mutagenic translesion synthesis within hprt during DNA replication and (2) lethality of mutant cells following replication fork breakage on the X chromosome, caused by unrepaired double-strand breaks or large deletions/translocations encompassing essential genes flanking hprt. These findings, along with studies showing increased spontaneous mutability of FA cells at two autosomal loci, support a model in which FA proteins promote both translesion synthesis at replication-blocking lesions and repair of broken replication forks by homologous recombination and DNA end joining. The essence of this model is that the FANC protein pathway serves to restrict the severity of mutational outcome by favoring base substitutions and small deletions over larger deletions and chromosomal rearrangements.

DNA Polymerase ζ is essential for hexavalent chromium-induced mutagenesis

PubMed Central

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

2009-01-01

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

Phenotypic heterogeneity in a bacteriophage population only appears as stress-induced mutagenesis.

PubMed

Yosef, Ido; Edgar, Rotem; Qimron, Udi

2016-11-01

Stress-induced mutagenesis has been studied in cancer cells, yeast, bacteria, and archaea, but not in viruses. In a recent publication, we present a bacteriophage model showing an apparent stress-induced mutagenesis. We show that the stress does not drive the mutagenesis, but only selects the fittest mutants. The mechanism underlying the observed phenomenon is a phenotypic heterogeneity that resembles persistence of the viral population. The new findings, the background for the ongoing debate on stress-induced mutagenesis, and the phenotypic heterogeneity underlying a novel phage infection strategy are discussed in this short manuscript.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans.

PubMed

Noma, Kentaro; Jin, Yishi

2016-11-14

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

mtDNA Mutagenesis Disrupts Pluripotent Stem Cell Function by Altering Redox Signaling

PubMed Central

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 associated with an increase in mitochondrial H2O2, reduced PSC reprogramming efficiency, and self-renewal. Mitochondria-targeted ubiquinone, MitoQ, and N-acetyl-L-cysteine efficiently rescued these defects, indicating that both reprogramming efficiency and stemness are modified by mitochondrial ROS. The redox sensitivity, however, rendered PSCs and especially neural stem cells sensitive to MitoQ toxicity. Our results imply that stem cell compartment warrants special attention when the safety of new antioxidants is assessed and point to an essential role for mitochondrial redox signaling in maintaining normal stem cell function. PMID:26027936

Dominant-Negative Inhibition of Prion Formation Diminished by Deletion Mutagenesis of the Prion Protein

PubMed Central

Zulianello, Laurence; Kaneko, Kiyotoshi; Scott, Michael; Erpel, Susanne; Han, Dong; Cohen, Fred E.; Prusiner, Stanley B.

2000-01-01

Polymorphic basic residues near the C terminus of the prion protein (PrP) in humans and sheep appear to protect against prion disease. In heterozygotes, inhibition of prion formation appears to be dominant negative and has been simulated in cultured cells persistently infected with scrapie prions. The results of nuclear magnetic resonance and mutagenesis studies indicate that specific substitutions at the C-terminal residues 167, 171, 214, and 218 of PrPC act as dominant-negative, inhibitors of PrPSc formation (K. Kaneko et al., Proc. Natl. Acad. Sci. USA 94:10069–10074, 1997). Trafficking of substituted PrPC to caveaola-like domains or rafts by the glycolipid anchor was required for the dominant-negative phenotype; interestingly, amino acid replacements at multiple sites were less effective than single-residue substitutions. To elucidate which domains of PrPC are responsible for dominant-negative inhibition of PrPSc formation, we analyzed whether N-terminally truncated PrP(Q218K) molecules exhibited dominant-negative effects in the conversion of full-length PrPC to PrPSc. We found that the C-terminal domain of PrP is not sufficient to impede the conversion of the full-length PrPC molecule and that N-terminally truncated molecules (with residues 23 to 88 and 23 to 120 deleted) have reduced dominant-negative activity. Whether the N-terminal region of PrP acts by stabilizing the C-terminal domain of the molecule or by modulating the binding of PrPC to an auxiliary molecule that participates in PrPSc formation remains to be established. PMID:10756050

Stress-Induced Mutagenesis: Implications in Cancer and Drug Resistance.

PubMed

Fitzgerald, Devon M; Hastings, P J; Rosenberg, Susan M

2017-03-01

Genomic instability underlies many cancers and generates genetic variation that drives cancer initiation, progression, and therapy resistance. In contrast with classical assumptions that mutations occur purely stochastically at constant, gradual rates, microbes, plants, flies, and human cancer cells possess mechanisms of mutagenesis that are upregulated by stress responses. These generate transient, genetic-diversity bursts that can propel evolution, specifically when cells are poorly adapted to their environments-that is, when stressed. We review molecular mechanisms of stress-response-dependent (stress-induced) mutagenesis that occur from bacteria to cancer, and are activated by starvation, drugs, hypoxia, and other stressors. We discuss mutagenic DNA break repair in Escherichia coli as a model for mechanisms in cancers. The temporal regulation of mutagenesis by stress responses and spatial restriction in genomes are common themes across the tree of life. Both can accelerate evolution, including the evolution of cancers. We discuss possible anti-evolvability drugs, aimed at targeting mutagenesis and other variation generators, that could be used to delay the evolution of cancer progression and therapy resistance.

Stress-Induced Mutagenesis: Implications in Cancer and Drug Resistance

PubMed Central

Fitzgerald, Devon M.; Hastings, P.J.; Rosenberg, Susan M.

2017-01-01

Genomic instability underlies many cancers and generates genetic variation that drives cancer initiation, progression, and therapy resistance. In contrast with classical assumptions that mutations occur purely stochastically at constant, gradual rates, microbes, plants, flies, and human cancer cells possess mechanisms of mutagenesis that are upregulated by stress responses. These generate transient, genetic-diversity bursts that can propel evolution, specifically when cells are poorly adapted to their environments—that is, when stressed. We review molecular mechanisms of stress-response-dependent (stress-induced) mutagenesis that occur from bacteria to cancer, and are activated by starvation, drugs, hypoxia, and other stressors. We discuss mutagenic DNA break repair in Escherichia coli as a model for mechanisms in cancers. The temporal regulation of mutagenesis by stress responses and spatial restriction in genomes are common themes across the tree of life. Both can accelerate evolution, including the evolution of cancers. We discuss possible anti-evolvability drugs, aimed at targeting mutagenesis and other variation generators, that could be used to delay the evolution of cancer progression and therapy resistance. PMID:29399660

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

PubMed

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

2016-11-01

Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95 pdz3 ) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

A Practical Teaching Course in Directed Protein Evolution Using the Green Fluorescent Protein as a Model

ERIC Educational Resources Information Center

Ruller, Roberto; Silva-Rocha, Rafael; Silva, Artur; Schneider, Maria Paula Cruz; Ward, Richard John

2011-01-01

Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from "Aequorea victoria" by a random mutagenesis strategy using error-prone polymerase…

Protein and genome evolution in Mammalian cells for biotechnology applications.

PubMed

Majors, Brian S; Chiang, Gisela G; Betenbaugh, Michael J

2009-06-01

Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.

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

PubMed Central

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

2014-01-01

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

Role of drosophila in chemical mutagenesis testing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nix, C.E.; Brewen, B.

1978-01-01

An important question facing our society is the impact of numerous chemical insults on the health of man and his environment. Faced with a staggering array of chemicals and enormous testing costs, only a few chemicals can be tested for possible carcinogenic effects. Recent results with the Salmonella/mammalian microsome mutagenesis bioassay system demonstrate a striking correlation between carcinogenicity and mutagenicity of many chemical compounds and offer the possibility that mutagenesis assay systems can provide a quick identification of potential carcinogens. Results from microbial assays can serve as a guideline for further mutagenesis testing as well as identify those compounds requiringmore » more extensive analysis in mammalian systems. Reliance on the results from a single mutagenic assay system is rather risky. It would be preferable to use a battery of tests (the tier approach) which would include the rapid microbial assays as well as mammalian systems. Also the use of Drosophila as a bridge between the microbial and mammalian assays has many desirable features which are discussed.« less

Use of CRISPR/Cas Genome Editing Technology for Targeted Mutagenesis in Rice.

PubMed

Xu, Rongfang; Wei, Pengcheng; Yang, Jianbo

2017-01-01

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system is a newly emerging mutagenesis (gene-editing) tool in genetic engineering. Among the agriculturally important crops, several genes have been successfully mutated by the system, and some agronomic important traits have been rapidly generated, which indicates the potential applications in both scientific research and plant breeding. In this chapter, we describe a standard gene-editing procedure to effectively target rice genes and to make specific rice mutants using the CRISPR/Cas9 system mediated by Agrobacterium transformation.

Revised Mechanism and Improved Efficiency of the QuikChange Site-Directed Mutagenesis Method.

PubMed

Xia, Yongzhen; Xun, Luying

2017-01-01

Site-directed mutagenesis has been widely used for the substitution, addition or deletion of nucleotide residues in a defined DNA sequence. QuikChange™ site-directed mutagenesis and its related protocols have been widely used for this purpose because of convenience and efficiency. We have recently demonstrated that the mechanism of the QuikChange™ site-directed mutagenesis process is different from that being proposed. The new mechanism promotes the use of partially overlapping primers and commercial PCR enzymes for efficient PCR and mutagenesis.

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).

PubMed

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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

PubMed Central

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

2015-01-01

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

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs.

PubMed

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

2015-06-01

Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward. Copyright © 2015 by the Genetics Society of America.

p53 Mutagenesis by Benzo[a]pyrene derived Radical Cations

PubMed Central

Sen, Sushmita; Bhojnagarwala, Pratik; Francey, Lauren; Lu, Ding; Jeffrey Field, Trevor M. Penning

2013-01-01

Benzo[a]pyrene (B[a]P), a major human carcinogen in combustion products such as cigarette smoke and diesel exhaust, is metabolically activated into DNA-reactive metabolites via three different enzymatic pathways. The pathways are the anti-(+)-benzo[a]pyrene 7,8-diol 9, 10-epoxide pathway (P450/ epoxide hydrolase catalyzed) (B[a]PDE), the benzo[a]pyrene o-quinone pathway (aldo ketose reductase (AKR) catalyzed) and the B[a]P radical cation pathway (P450 peroxidase catalyzed). We used a yeast p53 mutagenesis system to assess mutagenesis by B[a]P radical cations. Because radical cations are short-lived, they were generated in situ by reacting B[a]P with cumene hydroperoxide (CuOOH) and horse radish peroxidase (HRP) and then monitoring the generation of the more stable downstream products, B[a]P-1,6-dione and B[a]P-3,6-dione. Based on the B[a]P-1,6 and 3,6-dione formation, approximately 4µM of radical cation was generated. In the mutagenesis assays, the radical cations produced in situ showed a dose-dependent increase in mutagenicity from 0.25 µM to 10 µM B[a]P with no significant increase seen with further escalation to 50 µM B[a]P. However, mutagenesis was 200-fold less than with the AKR pathway derived B[a]P, 7–8 dione. Mutant p53 plasmids, which yield red colonies, were recovered from the yeast to study the pattern and spectrum of mutations. The mutation pattern observed was G to T (31%) > G to C (29%) > G to A (14%). The frequency of codons mutated by the B[a]P radical cations was essentially random and not enriched at known cancer hotspots. The quinone products of radical cations, B[a]P-1,6-dione and B[a]P-3,6-dione were more mutagenic than the radical cation reactions, but still less mutagenic than AKR derived B[a]P-7,8-dione. We conclude that B[a]P radical cations and their quinone products are weakly mutagenic in this yeast-based system compared to redox cycling PAH o-quinones. PMID:22768918

History of the science of mutagenesis from a personal perspective.

PubMed

Malling, Heinrich V

2004-01-01

A career in the study of mutagenesis spanning 50 years is a gift few scientists have been bestowed. My tenure in the field started in 1953, the year the structure of DNA became known (Watson and Crick [1953]: Nature 171:737). Before that time, it was suspected that DNA was the genetic material based on the research of Oswald T. Avery (Avery et al. [1944]: J Exp Med 79:137), but many scientists still believed that proteins or polysaccharides could be the genetic material. The present article describes a lifetime of personal experience in the field of chemical mutagenesis. The methods used to treat viruses with chemical mutagens were well developed in the 1950s. Here I review the early use of nitrous acid and hydroxylamine as mutagens in eukaryotes, the development of methods for the metabolic activation of mutagens by microsomal preparations, and the selection of a mutant tester set for the qualitative characterization of the mutagenic activity of chemicals. These studies provided critical background information that was used by Bruce Ames in the development of his Salmonella/microsome assay, widely known as the Ames test (Ames et al. [1973]: Proc Nat Acad Sci USA 70:2281-2285). This article also describes how a set of diagnostic chemical mutagens was selected and used to identify the molecular nature of gene mutations. Today, DNA sequencing has replaced the use of diagnostic mutagens, but studies of this kind formed the foundation of modern mutation research. They also helped set the stage for the organization of the Environmental Mutagen Society and the Environmental Mutagen Information Center, which are described. The article ends with the development of mammalian single-cell mutation assays, the first system for studying in vivo mutagenesis using recoverable vectors in transgenic animals, other mutation assays in intact mammals, and my thoughts on the critically important area of germ cell mutagenesis. This narrative is not a complete autobiographical account

ENU mutagenesis to generate genetically modified rat models.

PubMed

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

2010-01-01

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

One-step random mutagenesis by error-prone rolling circle amplification

PubMed Central

Fujii, Ryota; Kitaoka, Motomitsu; Hayashi, Kiyoshi

2004-01-01

In vitro random mutagenesis is a powerful tool for altering properties of enzymes. We describe here a novel random mutagenesis method using rolling circle amplification, named error-prone RCA. This method consists of only one DNA amplification step followed by transformation of the host strain, without treatment with any restriction enzymes or DNA ligases, and results in a randomly mutated plasmid library with 3–4 mutations per kilobase. Specific primers or special equipment, such as a thermal-cycler, are not required. This method permits rapid preparation of randomly mutated plasmid libraries, enabling random mutagenesis to become a more commonly used technique. PMID:15507684

New mutations affecting induced mutagenesis in yeast.

PubMed

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

1985-01-01

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

Extinction of Zika virus and Usutu virus by lethal mutagenesis reveals different patterns of sensitivity to three mutagenic drugs.

PubMed

Bassi, Maria Rosaria; Sempere, Raquel Navarro; Meyn, Prashansa; Polacek, Charlotta; Arias, Armando

2018-06-18

Flaviviruses constitute an increasing source of public health concern with growing numbers of pathogens causing disease, and a geographic spread to temperate climates. Despite a large body of evidence supporting mutagenesis as a conceivable antiviral strategy, there is currently no data on the sensitivity to increased mutagenesis for Zika virus (ZIKV) and Usutu virus (USUV), two emerging flaviviral threats. In this study, we demonstrate that both viruses are sensitive to three ribonucleosides that have shown mutagenic activity against other RNA viruses - favipiravir, ribavirin and 5-fluorouracil - while they remain unaffected by a mutagenic deoxyribonucleoside. Serial cell culture passages of ZIKV in the presence of these compounds resulted in the rapid extinction of infectivity, suggesting elevated sensitivity to mutagenesis. USUV extinction was achieved when a 10-fold dilution was applied between every passage, but not in experiments involving undiluted virus, indicating an overall lower susceptibility than ZIKV. Although both viruses are inhibited by the same three drugs, ZIKV is relatively more susceptive to serial passage in the presence of purine analogues (favipiravir and ribavirin) while USUV replication is suppressed more efficiently by 5-fluorouracil. These differences in sensitivity typically correlate with the increases in the mutation frequencies observed in each nucleoside treatment. These results are relevant to the development of efficient therapies based on lethal mutagenesis, and support the rational selection of different mutagenic nucleosides for each pathogen. We will discuss the implications of these results to the fidelity of flavivirus replication, and the design of antiviral therapies based on lethal mutagenesis. Copyright © 2018 Bassi et al.

Mutagenesis of a bacteriophage lytic enzyme PlyGBS significantly increases its antibacterial activity against group B streptococci.

PubMed

Cheng, Qi; Fischetti, Vincent A

2007-04-01

Group B streptococci (GBS) are the leading cause of neonatal meningitis and sepsis worldwide. Intrapartum antibiotic prophylaxis (IAP) is the current prevention strategy given to pregnant women with confirmed vaginal GBS colonization. Due to antibiotic resistance identified in GBS, we previously developed another strategy using a bacteriophage lytic enzyme, PlyGBS, to reduce vaginal GBS colonization. In this study, various DNA mutagenesis methods were explored to produce PlyGBS mutants with increased lytic activity against GBS. Several hyperactive mutants were identified that contain only the endopeptidase domain found in the N-terminal region of PlyGBS and represent only about one-third of the wild-type PlyGBS in length. Significantly, these mutants not only have 18-28-fold increases in specific activities compared to PlyGBS, but they also have a similar activity spectrum against several streptococcal species. One of the hyperactive mutants, PlyGBS90-1, reduced the GBS colonization from >5 logs of growth per mouse to <50 colony-forming units (cfu) 4 h post treatment ( approximately 4-log reduction) using a single dose in a mouse vaginal model. A reduction in GBS colonization before delivery should significantly reduce neonatal GBS infection providing a safe alternative to IAP.

Roles of Salmonella typhimurium umuDC and samAB in UV mutagenesis and UV sensitivity.

PubMed Central

Nohmi, T; Yamada, M; Watanabe, M; Murayama, S Y; Sofuni, T

1992-01-01

Expression of the umuDC operon is required for UV mutagenesis and most chemical mutagenesis in Escherichia coli. The closely related species Salmonella typhimurium has two sets of umuDC-like operons; the samAB operon is located in a 60-MDa cryptic plasmid, while the S. typhimurium umuDC (umuDCST) operon resides in a chromosome. The roles of these two umuDC-like operons in UV mutagenesis and UV sensitivity of S. typhimurium were investigated. A pBR322-derived plasmid carrying the samAB operon more efficiently restored UV mutability to a umuD44 strain and a umuC122::Tn5 strain of E. coli than a plasmid carrying the umuDCST operon did. When the umuDCST operon was specifically deleted from the chromosome of S. typhimurium TA2659, the resulting strain was not UV mutable and was more sensitive to the killing effect of UV irradiation than the parent strain was. Curing of the 60-MDa cryptic plasmid carrying the samAB operon did not influence the UV mutability of strain TA2659 but did increase its resistance to UV killing. A pSC101-derived plasmid carrying the samAB operon did not restore UV mutability to a umuD44 strain of E. coli, whereas pBR322- or pBluescript-derived plasmids carrying the samAB operon efficiently did restore UV mutability. We concluded that the umuDCST operon plays a major role in UV mutagenesis in S. typhimurium and that the ability of the samAB operon to promote UV mutagenesis is strongly affected by gene dosage. Possible reasons for the poor ability of samAB to promote UV mutagenesis when it is present on low-copy-number plasmids are discussed. Images PMID:1400244

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

PubMed

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

2006-02-01

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

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

PubMed Central

Zarei, Omid; Hamzeh-Mivehroud, Maryam; Benvenuti, Silvia; Ustun-Alkan, Fulya; Dastmalchi, Siavoush

2017-01-01

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. PMID:28507948

CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

PubMed

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

2014-09-05

Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila. Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency. Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. Furthermore, by including multiple guide RNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. The CRISPR/Cas9-mediated conditional mutagenesis system provides a simple and effective tool for gene function analysis, and complements the existing RNAi approach. Copyright © 2014 Xue et al.

Engineering Signal Peptides for Enhanced Protein Secretion from Lactococcus lactis

PubMed Central

Ng, Daphne T. W.

2013-01-01

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts. PMID:23124224

Engineering signal peptides for enhanced protein secretion from Lactococcus lactis.

PubMed

Ng, Daphne T W; Sarkar, Casim A

2013-01-01

Lactococcus lactis is an attractive vehicle for biotechnological production of proteins and clinical delivery of therapeutics. In many such applications using this host, it is desirable to maximize secretion of recombinant proteins into the extracellular space, which is typically achieved by using the native signal peptide from a major secreted lactococcal protein, Usp45. In order to further increase protein secretion from L. lactis, inherent limitations of the Usp45 signal peptide (Usp45sp) must be elucidated. Here, we performed extensive mutagenesis on Usp45sp to probe the effects of both the mRNA sequence (silent mutations) and the peptide sequence (amino acid substitutions) on secretion. We screened signal peptides based on their resulting secretion levels of Staphylococcus aureus nuclease and further evaluated them for secretion of Bacillus subtilis α-amylase. Silent mutations alone gave an increase of up to 16% in the secretion of α-amylase through a mechanism consistent with relaxed mRNA folding around the ribosome binding site and enhanced translation. Targeted amino acid mutagenesis in Usp45sp, combined with additional silent mutations from the best clone in the initial screen, yielded an increase of up to 51% in maximum secretion of α-amylase while maintaining secretion at lower induction levels. The best sequence from our screen preserves the tripartite structure of the native signal peptide but increases the positive charge of the n-region. Our study presents the first example of an engineered L. lactis signal peptide with a higher secretion yield than Usp45sp and, more generally, provides strategies for further enhancing protein secretion in bacterial hosts.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Demple, Bruce

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, cellmore » 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.« less

Development of potent in vivo mutagenesis plasmids with broad mutational spectra

PubMed Central

Badran, Ahmed H.; Liu, David R.

2015-01-01

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

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

PubMed

Badran, Ahmed H; Liu, David R

2015-10-07

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

Genetically modified proteins: functional improvement and chimeragenesis

PubMed Central

Balabanova, Larissa; Golotin, Vasily; Podvolotskaya, Anna; Rasskazov, Valery

2015-01-01

This review focuses on the emerging role of site-specific mutagenesis and chimeragenesis for the functional improvement of proteins in areas where traditional protein engineering methods have been extensively used and practically exhausted. The novel path for the creation of the novel proteins has been created on the farther development of the new structure and sequence optimization algorithms for generating and designing the accurate structure models in result of x-ray crystallography studies of a lot of proteins and their mutant forms. Artificial genetic modifications aim to expand nature's repertoire of biomolecules. One of the most exciting potential results of mutagenesis or chimeragenesis finding could be design of effective diagnostics, bio-therapeutics and biocatalysts. A sampling of recent examples is listed below for the in vivo and in vitro genetically improvement of various binding protein and enzyme functions, with references for more in-depth study provided for the reader's benefit. PMID:26211369

Simple-MSSM: a simple and efficient method for simultaneous multi-site saturation mutagenesis.

PubMed

Cheng, Feng; Xu, Jian-Miao; Xiang, Chao; Liu, Zhi-Qiang; Zhao, Li-Qing; Zheng, Yu-Guo

2017-04-01

To develop a practically simple and robust multi-site saturation mutagenesis (MSSM) method that enables simultaneously recombination of amino acid positions for focused mutant library generation. A general restriction enzyme-free and ligase-free MSSM method (Simple-MSSM) based on prolonged overlap extension PCR (POE-PCR) and Simple Cloning techniques. As a proof of principle of Simple-MSSM, the gene of eGFP (enhanced green fluorescent protein) was used as a template gene for simultaneous mutagenesis of five codons. Forty-eight randomly selected clones were sequenced. Sequencing revealed that all the 48 clones showed at least one mutant codon (mutation efficiency = 100%), and 46 out of the 48 clones had mutations at all the five codons. The obtained diversities at these five codons are 27, 24, 26, 26 and 22, respectively, which correspond to 84, 75, 81, 81, 69% of the theoretical diversity offered by NNK-degeneration (32 codons; NNK, K = T or G). The enzyme-free Simple-MSSM method can simultaneously and efficiently saturate five codons within one day, and therefore avoid missing interactions between residues in interacting amino acid networks.

Low-dose radiation attenuates chemical mutagenesis in vivo.

PubMed

Kakinuma, Shizuko; Yamauchi, Kazumi; Amasaki, Yoshiko; Nishimura, Mayumi; Shimada, Yoshiya

2009-09-01

The biological effects of low-dose radiation are not only of social concern but also of scientific interest. The radioadaptive response, which is defined as an increased radioresistance by prior exposure to low-dose radiation, has been extensively studied both in vitro and in vivo. Here we briefly review the radioadaptive response with respect to mutagenesis, survival rate, and carcinogenesis in vivo, and introduce our recent findings of cross adaptation in mouse thymic cells, that is, the suppressive effect of repeated low-dose radiation on mutation induction by the alkylating agent N-ethyl-N-nitrosourea.

Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA.

PubMed

Kanazashi, Yuhei; Hirose, Aya; Takahashi, Ippei; Mikami, Masafumi; Endo, Masaki; Hirose, Sakiko; Toki, Seiichi; Kaga, Akito; Naito, Ken; Ishimoto, Masao; Abe, Jun; Yamada, Tetsuya

2018-03-01

Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T 2 seeds. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD). Most of the T 1 plants had heterozygous and/or chimeric mutations for the targeted loci. The sequencing analysis of T 1 and T 2 generations indicates that putative mutation induced in the T 0 plant is transmitted to the T 1 generation. The inheritable mutation induced in the T 1 plant was also detected. This result indicates that continuous induction of mutations during T 1 plant development increases the occurrence of mutations in germ cells, which ensures the transmission of mutations to the next generation. Simultaneous site-directed mutagenesis in both GmPPD loci was confirmed in at least 33% of T 2 seeds examined. Approximately 19% of double mutants did not contain the Cas9/gRNA expression construct. Double mutants with frameshift mutations in both GmPPD1 and GmPPD2 had dome-shaped trifoliate leaves, extremely twisted pods, and produced few seeds. Taken together, our data indicate that continuous induction of mutations in the whole plant and advancing generations of transgenic plants enable efficient simultaneous site-directed mutagenesis in duplicated loci in soybean.

UVB-induced mutagenesis in hairless {lambda}lacZ-transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

UVB-induced mutagenesis was studied in hairless 40.6 transgenic mice (Muta{trademark}Mouse), which contain the {lambda}gt1OlacZ shuttle vector as a target for mutagenesis. Mice were exposed at the dorsal side to either single doses of 200, 500, 800, or 1000 J/m{sup 2} UVB or to two successive irradiations of either 200 and 800 J/m{sup 2} UVB, with intervals of 1,3, or 5 days, or to 800 and 200 J/m{sup 2} UVB with a 5-day interval. At 23 days after the last exposure, lacZ mutant frequencies (MF) were determined in the epidermis. The lacZ MF increased linearly with increasing dose of UVB. Themore » mutagenic effect of two successive irradiations appeared to be additive. The UV-induced mutation spectrum was dominated by G:C{r_arrow}A:T transitions at dipyrimidine sites. DNA-sequence analysis of spontaneously mutated phages showed a diverse spectrum consisting of insertions, deletions and G:C {r_arrow} A:T transitions at CpG sites. the results indicate that the hairless {lambda}lacZ-transgenic mouse is a suitable in vivo model for studying UVB-induced mutations. 29 refs., 5 tabs.« less

Saturation Mutagenesis of Burkholderia cepacia R34 2,4-Dinitrotoluene Dioxygenase at DntAc Valine 350 for Synthesizing Nitrohydroquinone, Methylhydroquinone, and Methoxyhydroquinone

PubMed Central

Keenan, Brendan G.; Leungsakul, Thammajun; Smets, Barth F.; Wood, Thomas K.

2004-01-01

Saturation mutagenesis of the 2,4-dinitrotoluene dioxygenase (DDO) of Burkholderia cepacia R34 at position valine 350 of the DntAc α-subunit generated mutant V350F with significantly increased activity towards o-nitrophenol (47 times), m-nitrophenol (34 times), and o-methoxyphenol (174 times) as well as an expanded substrate range that now includes m-methoxyphenol, o-cresol, and m-cresol (wild-type DDO had no detectable activity for these substrates). Another mutant, V350M, also displays increased activity towards o-nitrophenol (20 times) and o-methoxyphenol (162 times) as well as novel activity towards o-cresol. Products were synthesized using whole Escherichia coli TG1 cells expressing the recombinant R34 dntA loci from pBS(Kan)R34, and the initial rates of product formation were determined at 1 mM substrate by reverse-phase high-pressure liquid chromatography. V350F produced both nitrohydroquinone at a rate of 0.75 ± 0.15 nmol/min/mg of protein and 3-nitrocatechol at a rate of 0.069 ± 0.001 nmol/min/mg of protein from o-nitrophenol, 4-nitrocatechol from m-nitrophenol at 0.29 ± 0.02 nmol/min/mg of protein, methoxyhydroquinone from o-methoxyphenol at 2.5 ± 0.6 nmol/min/mg of protein, methoxyhydroquinone from m-methoxyphenol at 0.55 ± 0.02 nmol/min/mg of protein, both methylhydroquinone at 1.52 ± 0.02 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.74 ± 0.05 nmol/min/mg of protein from o-cresol, and methylhydroquinone at 0.43 ± 0.1 nmol/min/mg of protein from m-cresol. V350M produced both nitrohydroquinone at a rate of 0.33 nmol/min/mg of protein and 3-nitrocatechol at 0.089 nmol/min/mg of protein from o-nitrophenol, methoxyhydroquinone from o-methoxyphenol at 2.4 nmol/min/mg of protein, methylhydroquinone at 1.97 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.11 nmol/min/mg of protein from o-cresol. The DDO variants V350F and V350M also exhibited 10-fold-enhanced activity towards naphthalene (8 ± 2.6 nmol/min/mg of protein), forming (1R

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

PubMed Central

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

2016-01-01

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

Increasing protein stability by improving beta-turns.

PubMed

Fu, Hailong; Grimsley, Gerald R; Razvi, Abbas; Scholtz, J Martin; Pace, C Nick

2009-11-15

Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability. 2009 Wiley-Liss, Inc.

INCREASING PROTEIN STABILITY BY IMPROVING BETA-TURNS

PubMed Central

Fu, Hailong; Grimsley, Gerald R.; Razvi, Abbas; Scholtz, J. Martin; Pace, C. Nick

2009-01-01

Our goal was to gain a better understanding of how protein stability can be increased by improving β-turns. We studied 22 β-turns in nine proteins with 66 to 370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some β-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein (HPr), Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase α-subunit (TSα), and Maltose binding protein (MBP). Of the fifteen single proline mutations, 11increased stability (Average = 0.8 ± 0.3; Range = 0.3 – 1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. Based on this and our previous work, we conclude that proteins can generally be stabilized by replacing non-proline residues with proline residues at the i + 1 position of Type I and II β-turns and at the i position in Type II β-turns. Other turn positions can sometimes be used if the φ angle is near −60° for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in β-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in β-turns that could be replaced by Gly to increase protein stability. Improving β-turns by substituting Pro residues is a generally useful way of increasing protein stability. PMID:19626709

Enhancement of protein production via the strong DIT1 terminator and two RNA-binding proteins in Saccharomyces cerevisiae.

PubMed

Ito, Yoichiro; Kitagawa, Takao; Yamanishi, Mamoru; Katahira, Satoshi; Izawa, Shingo; Irie, Kenji; Furutani-Seiki, Makoto; Matsuyama, Takashi

2016-11-15

Post-transcriptional upregulation is an effective way to increase the expression of transgenes and thus maximize the yields of target chemicals from metabolically engineered organisms. Refractory elements in the 3' untranslated region (UTR) that increase mRNA half-life might be available. In Saccharomyces cerevisiae, several terminator regions have shown activity in increasing the production of proteins by upstream coding genes; among these terminators the DIT1 terminator has the highest activity. Here, we found in Saccharomyces cerevisiae that two resident trans-acting RNA-binding proteins (Nab6p and Pap1p) enhance the activity of the DIT1 terminator through the cis element GUUCG/U within the 3'-UTR. These two RNA-binding proteins could upregulate a battery of cell-wall-related genes. Mutagenesis of the DIT1 terminator improved its activity by a maximum of 500% of that of the standard PGK1 terminator. Further understanding and improvement of this system will facilitate inexpensive and stable production of complicated organism-derived drugs worldwide.

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

PubMed

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

2017-06-22

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

Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

PubMed Central

Cozens, Christopher

2018-01-01

Abstract Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations – particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0.25% of total population) and which takes a single working day from purified plasmid to library transformation. We demonstrate its efficacy with whole gene codon reassignment of chloramphenicol acetyl transferase, mutating 19 codons in a single reaction in KOD DNA polymerase and generating high quality, multiple-site libraries in T7 RNA polymerase and Tgo DNA polymerase. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation. PMID:29409059

Roles for the yeast RAD18 and RAD52 DNA repair genes in UV mutagenesis.

PubMed

Armstrong, J D; Chadee, D N; Kunz, B A

1994-11-01

Experimental evidence indicates that although the Saccharomyces cerevisiae RAD18 and RAD52 genes are not required for nucleotide excision repair, they function in the processing of UV-induced DNA damage in yeast. Conflicting statements regarding the UV mutability of strains deleted for RAD18 prompted us to re-examine the influence of RAD18, and RAD52, on UV mutagenesis. To do so, we characterized mutations induced by UV in SUP4-o, a yeast suppressor tRNA gene. SUP4-o was maintained on a plasmid in isogenic strains that either carried one of two different rad18 deletions (rad18 delta) or had RAD52 disrupted. Both rad18 deletions decreased the frequency of UV-induced SUP4-o mutations to levels close to those for spontaneous mutagenesis in the rad18 delta backgrounds, and prevented a net increase in mutant yield. A detailed analysis of mutations isolated after UV irradiation of one of the rad18 delta strains uncovered little evidence of the specificity features typical for UV mutagenesis in the isogenic repair-proficient (RAD) parent (e.g., predominance of G.C-->A.T transitions). Evidently, UV induction of SUP4-o mutations is highly dependent on the RAD18 gene. Compared to the RAD strain, disruption of RAD52 reduced the frequency and yield of UV mutagenesis by about two-thirds. Closer inspection revealed that 80% of this reduction was due to a decrease in the frequency of G.C-->A.T transitions. In addition, there were differences in the distributions and site specificities of single base-pair substitutions. Thus, RAD52 also participates in UV mutagenesis of a plasmid-borne gene in yeast, but to a lesser extent than RAD18.

A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

PubMed

Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

1996-01-01

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

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

PubMed

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

2016-09-01

Whole-genome sequencing is a useful approach for identification of chemical-induced lesions, but previous applications involved tedious genetic mapping to pinpoint the causative mutations. We propose that saturation mutagenesis under low mutagenic loads, followed by whole-genome sequencing, should allow direct implication of genes by identifying multiple independent alleles of each relevant gene. We tested the hypothesis by performing three genetic screens with chemical mutagenesis in the social soil amoeba Dictyostelium discoideum Through genome sequencing, we successfully identified mutant genes with multiple alleles in near-saturation screens, including resistance to intense illumination and strong suppressors of defects in an allorecognition pathway. We tested the causality of the mutations by comparison to published data and by direct complementation tests, finding both dominant and recessive causative mutations. Therefore, our strategy provides a cost- and time-efficient approach to gene discovery by integrating chemical mutagenesis and whole-genome sequencing. The method should be applicable to many microbial systems, and it is expected to revolutionize the field of functional genomics in Dictyostelium by greatly expanding the mutation spectrum relative to other common mutagenesis methods. © 2016 Li et al.; Published by Cold Spring Harbor Laboratory Press.

Identification of essential genes in Streptococcus pneumoniae by allelic replacement mutagenesis.

PubMed

Song, Jae-Hoon; Ko, Kwan Soo; Lee, Ji-Young; Baek, Jin Yang; Oh, Won Sup; Yoon, Ha Sik; Jeong, Jin-Yong; Chun, Jongsik

2005-06-30

To find potential targets of novel antimicrobial agents, we identified essential genes of Streptococcus pneumoniae using comparative genomics and allelic replacement mutagenesis. We compared the genome of S. pneumoniae R6 with those of Bacillus subtilis, Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus, and selected 693 candidate target genes with > 40% amino acid sequence identity to the corresponding genes in at least two of the other species. The 693 genes were disrupted and 133 were found to be essential for growth. Of these, 32 encoded proteins of unknown function, and we were able to identify orthologues of 22 of these genes by genomic comparisons. The experimental method used in this study is easy to perform, rapid and efficient for identifying essential genes of bacterial pathogens.

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

PubMed

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

2013-11-19

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

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

PubMed Central

Peterson, A R; Peterson, H

1982-01-01

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

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

PubMed

Peterson, A R; Peterson, H

1982-03-01

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

PERMutation Using Transposase Engineering (PERMUTE): A Simple Approach for Constructing Circularly Permuted Protein Libraries.

PubMed

Jones, Alicia M; Atkinson, Joshua T; Silberg, Jonathan J

2017-01-01

Rearrangements that alter the order of a protein's sequence are used in the lab to study protein folding, improve activity, and build molecular switches. One of the simplest ways to rearrange a protein sequence is through random circular permutation, where native protein termini are linked together and new termini are created elsewhere through random backbone fission. Transposase mutagenesis has emerged as a simple way to generate libraries encoding different circularly permuted variants of proteins. With this approach, a synthetic transposon (called a permuteposon) is randomly inserted throughout a circularized gene to generate vectors that express different permuted variants of a protein. In this chapter, we outline the protocol for constructing combinatorial libraries of circularly permuted proteins using transposase mutagenesis, and we describe the different permuteposons that have been developed to facilitate library construction.

Directed mutagenesis of the Rickettsia prowazekii pld gene encoding phospholipase D.

PubMed

Driskell, Lonnie O; Yu, Xue-jie; Zhang, Lihong; Liu, Yan; Popov, Vsevolod L; Walker, David H; Tucker, Aimee M; Wood, David O

2009-08-01

Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligately intracytoplasmic bacterium, a lifestyle that imposes significant barriers to genetic manipulation. The key to understanding how this unique bacterium evades host immunity is the mutagenesis of selected genes hypothesized to be involved in virulence. The R. prowazekii pld gene, encoding a protein with phospholipase D activity, has been associated with phagosomal escape. To demonstrate the feasibility of site-directed knockout mutagenesis of rickettsial genes and to generate a nonrevertible vaccine strain, we utilized homologous recombination to generate a pld mutant of the virulent R. prowazekii strain Madrid Evir. Using linear DNA for transformation, a double-crossover event resulted in the replacement of the rickettsial wild-type gene with a partially deleted pld gene. Linear DNA was used to prevent potentially revertible single-crossover events resulting in plasmid insertion. Southern blot and PCR analyses were used to confirm the presence of the desired mutation and to demonstrate clonality. While no phenotypic differences were observed between the mutant and wild-type strains when grown in tissue culture, the pld mutant exhibited attenuated virulence in the guinea pig model. In addition, animals immunized with the mutant strain were protected against subsequent challenge with the virulent Breinl strain, suggesting that this transformant could serve as a nonrevertible, attenuated vaccine strain. This study demonstrates the feasibility of generating site-directed rickettsial gene mutants, providing a new tool for understanding rickettsial biology and furthering advances in the prevention of epidemic typhus.

GERM-LINE SPECIFIC FACTORS IN CHEMICAL MUTAGENESIS

EPA Science Inventory

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

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

PubMed Central

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

2017-01-01

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

Increased protein intake in military special operations.

PubMed

Ferrando, Arny A

2013-11-01

Special operations are so designated for the specialized military missions they address. As a result, special operations present some unique metabolic challenges. In particular, soldiers often operate in a negative energy balance in stressful and demanding conditions with little opportunity for rest or recovery. In this framework, findings inferred from the performance literature suggest that increased protein intake may be beneficial. In particular, increased protein intake during negative caloric balance maintains lean body mass and blood glucose production. The addition of protein to mixed macronutrient supplements is beneficial for muscle endurance and power endpoints, and the use of amino acids improves gross and fine motor skills. Increasing protein intake during periods of intense training and/or metabolic demand improves subsequent performance, improves muscular recovery, and reduces symptoms of psychological stress. Consumption of protein before sleep confers the anabolic responses required for the maintenance of lean mass and muscle recovery. A maximal response in muscle protein synthesis is achieved with the consumption of 20-25 g of protein alone. However, higher protein intakes in the context of mixed-nutrient ingestion also confer anabolic benefits by reducing protein breakdown. Restricted rations issued to special operators provide less than the RDA for protein ( ∼ 0.6 g/kg), and these soldiers often rely on commercial products to augment their rations. The provision of reasonable alternatives and/or certification of approved supplements by the U.S. Department of Defense would be prudent.

Differential involvement of the related DNA helicases Pif1p and Rrm3p in mtDNA point mutagenesis and stability.

PubMed

O'Rourke, Thomas W; Doudican, Nicole A; Zhang, Hong; Eaton, Jana S; Doetsch, Paul W; Shadel, Gerald S

2005-07-18

With the exception of base excision repair, conserved pathways and mechanisms that maintain mitochondrial genome stability have remained largely undelineated. In the budding yeast, Saccharomyces cerevisiae, Pif1p is a unique DNA helicase that is localized both to the nucleus and mitochondria, where it is involved in maintaining DNA integrity. We previously elucidated a role for Pif1p in oxidative mtDNA damage resistance that appears to be distinct from its postulated function in mtDNA recombination. Strains lacking Pif1p (pif1Delta) exhibit an increased rate of formation of petite mutants (an indicator of mtDNA instability) and elevated mtDNA point mutagenesis. Here we show that deletion of the RRM3 gene, which encodes a DNA helicase closely related to Pif1p, significantly rescues the petite-induction phenotype of a pif1Delta strain. However, suppression of this phenotype was not accompanied by a corresponding decrease in mtDNA point mutagenesis. Instead, deletion of RRM3 alone resulted in an increase in mtDNA point mutagenesis that was synergistic with that caused by a pif1Delta mutation. In addition, we found that over-expression of RNR1, encoding a large subunit of ribonucleotide reductase (RNR), rescued the petite-induction phenotype of a pif1Delta mutation to a similar extent as deletion of RRM3. This, coupled to our finding that the Rad53p protein kinase is phosphorylated in the rrm3Delta pif1Delta double-mutant strain, leads us to conclude that one mechanism whereby deletion of RRM3 influences mtDNA stability is by modulating mitochondrial deoxynucleoside triphosphate pools. We propose that this is accomplished by signaling through the conserved Mec1/Rad53, S-phase checkpoint pathway to induce the expression and activity of RNR. Altogether, our results define a novel role for Rrm3p in mitochondrial function and indicate that Pif1p and Rrm3p influence a common process (or processes) involved in mtDNA replication, repair, or stability.

Specificity protein, Sp1-mediated increased expression of Prdx6 as a curcumin-induced antioxidant defense in lens epithelial cells against oxidative stress

PubMed Central

Chhunchha, B; Fatma, N; Bhargavan, B; Kubo, E; Kumar, A; Singh, D P

2011-01-01

Peroxiredoxin 6 (Prdx6) is a pleiotropic oxidative stress-response protein that defends cells against reactive oxygen species (ROS)-induced damage. Curcumin, a naturally occurring agent, has diversified beneficial roles including cytoprotection. Using human lens epithelial cells (hLECs) and Prdx6-deficient cells, we show the evidence that curcumin protects cells by upregulating Prdx6 transcription via invoking specificity protein 1 (Sp1) activity against proapoptotic stimuli. Curcumin enhanced Sp1 and Prdx6 mRNA and protein expression in a concentration-dependent manner, as evidenced by western and real-time PCR analyses, and thereby negatively regulated ROS-mediated apoptosis by blunting ROS expression and lipid peroxidation. Bioinformatic analysis and DNA–protein binding assays disclosed three active Sp1 sites (−19/27, −61/69 and −82/89) in Prdx6 promoter. Co-transfection experiments with Sp1 and Prdx6 promoter–chloramphenicol acetyltransferase (CAT) constructs showed that CAT activity was dramatically increased in LECs or Sp1-deficient cells (SL2). Curcumin treatment of LECs enhanced Sp1 binding to its sites, consistent with curcumin-dependent stimulation of Prdx6 promoter with Sp1 sites and cytoprotection. Notably, disruption of Sp1 sites by point mutagenesis abolished curcumin transactivation of Prdx6. Also, curcumin failed to activate Prdx6 expression in the presence of Sp1 inhibitors, demonstrating that curcumin-mediated increased expression of Prdx6 was dependent on Sp1 activity. Collectively, the study may provide a foundation for developing transcription-based inductive therapy to reinforce endogenous antioxidant defense by using dietary supplements. PMID:22113199

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

PubMed Central

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

2011-01-01

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

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

PubMed

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

2015-06-01

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

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

PubMed

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

2013-11-13

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

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

PubMed

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

2016-01-01

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

Highly Efficient Targeted Mutagenesis in Mice Using TALENs

PubMed Central

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

2013-01-01

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

Enhancement of Schizochytrium DHA synthesis by plasma mutagenesis aided with malonic acid and zeocin screening.

PubMed

Zhao, Ben; Li, Yafei; Li, Changling; Yang, Hailin; Wang, Wu

2018-03-01

Schizochytrium sp. accumulates valuable polyunsaturated fatty acid (PUFA), such as docosahexaenoic acid (DHA). In order to increase DHA synthesis in this microorganism, physical or chemical mutagenesis aided with powerful screening methods are still preferable, as its DHA synthetic pathway has not yet been clearly defined for gene manipulation. To breed this agglomerate microorganism of thick cell wall and rather large genome for increasing lipid content and DHA percentage, a novel strategy of atmospheric and room temperature plasma (ARTP) mutagenesis coupled with stepped malonic acid (MA) and zeocin resistance screening was developed. The final resulted mutant strain mz-17 was selected with 1.8-fold increased DHA production. Accompanied with supplementation of Fe 2+ in shake flask cultivation, DHA production of 14.0 g/L on average was achieved. This work suggests that ARTP mutation combined with stepped MA and zeocin resistance screening is an efficient method of breeding Schizochytrium sp. of high DHA production, and might be applied on other microorganisms for obtaining higher desired PUFA products.

CHALLENGES FOR THE FUTURE IN ENVIRONMENTAL MUTAGENESIS

EPA Science Inventory

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

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

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

PubMed

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

2015-07-01

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

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

PubMed

Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

2015-08-01

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

Targeted mutagenesis in sea urchin embryos using TALENs.

PubMed

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

2014-01-01

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

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

PubMed Central

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

2015-01-01

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

News from the protein mutability landscape.

PubMed

Hecht, Maximilian; Bromberg, Yana; Rost, Burkhard

2013-11-01

Some mutations of protein residues matter more than others, and these are often conserved evolutionarily. The explosion of deep sequencing and genotyping increasingly requires the distinction between effect and neutral variants. The simplest approach predicts all mutations of conserved residues to have an effect; however, this works poorly, at best. Many computational tools that are optimized to predict the impact of point mutations provide more detail. Here, we expand the perspective from the view of single variants to the level of sketching the entire mutability landscape. This landscape is defined by the impact of substituting every residue at each position in a protein by each of the 19 non-native amino acids. We review some of the powerful conclusions about protein function, stability and their robustness to mutation that can be drawn from such an analysis. Large-scale experimental and computational mutagenesis experiments are increasingly furthering our understanding of protein function and of the genotype-phenotype associations. We also discuss how these can be used to improve predictions of protein function and pathogenicity of missense variants. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

Construction of a horseradish peroxidase resistant toward hydrogen peroxide by saturation mutagenesis.

PubMed

Asad, Sedigheh; Dastgheib, Seyed Mohammad Mehdi; Khajeh, Khosro

2016-11-01

Horseradish peroxidase (HRP) with a variety of potential biotechnological applications is still isolated from the horseradish root as a mixture of different isoenzymes with different biochemical properties. There is an increasing demand for preparations of high amounts of pure enzyme but its recombinant production is limited because of the lack of glycosylation in Escherichia coli and different glycosylation patterns in yeasts which affects its stability parameters. The goal of this study was to increase the stability of non-glycosylated enzyme, which is produced in E. coli, toward hydrogen peroxide via mutagenesis. Asparagine 268, one of the N-glycosylation sites of the enzyme, has been mutated via saturation mutagenesis using the megaprimer method. Modification and miniaturization of previously described protocols enabled screening of a library propagated in E. coli XJb (DE3). The library of mutants was screened for stability toward hydrogen peroxide with azinobis (ethylbenzthiazoline sulfonate) as a reducing substrate. Asn268Gly mutant, the top variant from the screening, exhibited 18-fold increased stability toward hydrogen peroxide and twice improved thermal stability compared with the recombinant HRP. Moreover, the substitution led to 2.5-fold improvement in the catalytic efficiency with phenol/4-aminoantipyrine. Constructed mutant represents a stable biocatalyst, which may find use in medical diagnostics, biosensing, and bioprocesses. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Faux Mutagenesis: Teaching Troubleshooting through Controlled Failure

ERIC Educational Resources Information Center

Hartberg, Yasha

2006-01-01

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

The DNA cytosine deaminase APOBEC3H haplotype I likely contributes to breast and lung cancer mutagenesis.

PubMed

Starrett, Gabriel J; Luengas, Elizabeth M; McCann, Jennifer L; Ebrahimi, Diako; Temiz, Nuri A; Love, Robin P; Feng, Yuqing; Adolph, Madison B; Chelico, Linda; Law, Emily K; Carpenter, Michael A; Harris, Reuben S

2016-09-21

Cytosine mutations within TCA/T motifs are common in cancer. A likely cause is the DNA cytosine deaminase APOBEC3B (A3B). However, A3B-null breast tumours still have this mutational bias. Here we show that APOBEC3H haplotype I (A3H-I) provides a likely solution to this paradox. A3B-null tumours with this mutational bias have at least one copy of A3H-I despite little genetic linkage between these genes. Although deemed inactive previously, A3H-I has robust activity in biochemical and cellular assays, similar to A3H-II after compensation for lower protein expression levels. Gly105 in A3H-I (versus Arg105 in A3H-II) results in lower protein expression levels and increased nuclear localization, providing a mechanism for accessing genomic DNA. A3H-I also associates with clonal TCA/T-biased mutations in lung adenocarcinoma suggesting this enzyme makes broader contributions to cancer mutagenesis. These studies combine to suggest that A3B and A3H-I, together, explain the bulk of 'APOBEC signature' mutations in cancer.

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

PubMed

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

2018-03-30

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

Enhancement of protein production via the strong DIT1 terminator and two RNA-binding proteins in Saccharomyces cerevisiae

PubMed Central

Ito, Yoichiro; Kitagawa, Takao; Yamanishi, Mamoru; Katahira, Satoshi; Izawa, Shingo; Irie, Kenji; Furutani-Seiki, Makoto; Matsuyama, Takashi

2016-01-01

Post-transcriptional upregulation is an effective way to increase the expression of transgenes and thus maximize the yields of target chemicals from metabolically engineered organisms. Refractory elements in the 3′ untranslated region (UTR) that increase mRNA half-life might be available. In Saccharomyces cerevisiae, several terminator regions have shown activity in increasing the production of proteins by upstream coding genes; among these terminators the DIT1 terminator has the highest activity. Here, we found in Saccharomyces cerevisiae that two resident trans-acting RNA-binding proteins (Nab6p and Pap1p) enhance the activity of the DIT1 terminator through the cis element GUUCG/U within the 3′-UTR. These two RNA-binding proteins could upregulate a battery of cell-wall–related genes. Mutagenesis of the DIT1 terminator improved its activity by a maximum of 500% of that of the standard PGK1 terminator. Further understanding and improvement of this system will facilitate inexpensive and stable production of complicated organism-derived drugs worldwide. PMID:27845367

Fragile DNA Motifs Trigger Mutagenesis at Distant Chromosomal Loci in Saccharomyces cerevisiae

PubMed Central

Saini, Natalie; Zhang, Yu; Nishida, Yuri; Sheng, Ziwei; Choudhury, Shilpa; Mieczkowski, Piotr; Lobachev, Kirill S.

2013-01-01

DNA sequences capable of adopting non-canonical secondary structures have been associated with gross-chromosomal rearrangements in humans and model organisms. Previously, we have shown that long inverted repeats that form hairpin and cruciform structures and triplex-forming GAA/TTC repeats induce the formation of double-strand breaks which trigger genome instability in yeast. In this study, we demonstrate that breakage at both inverted repeats and GAA/TTC repeats is augmented by defects in DNA replication. Increased fragility is associated with increased mutation levels in the reporter genes located as far as 8 kb from both sides of the repeats. The increase in mutations was dependent on the presence of inverted or GAA/TTC repeats and activity of the translesion polymerase Polζ. Mutagenesis induced by inverted repeats also required Sae2 which opens hairpin-capped breaks and initiates end resection. The amount of breakage at the repeats is an important determinant of mutations as a perfect palindromic sequence with inherently increased fragility was also found to elevate mutation rates even in replication-proficient strains. We hypothesize that the underlying mechanism for mutagenesis induced by fragile motifs involves the formation of long single-stranded regions in the broken chromosome, invasion of the undamaged sister chromatid for repair, and faulty DNA synthesis employing Polζ. These data demonstrate that repeat-mediated breaks pose a dual threat to eukaryotic genome integrity by inducing chromosomal aberrations as well as mutations in flanking genes. PMID:23785298

Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion.

PubMed

Fulton, Benjamin O; Sachs, David; Schwarz, Megan C; Palese, Peter; Evans, Matthew J

2017-08-01

The molecular constraints affecting Zika virus (ZIKV) evolution are not well understood. To investigate ZIKV genetic flexibility, we used transposon mutagenesis to add 15-nucleotide insertions throughout the ZIKV MR766 genome and subsequently deep sequenced the viable mutants. Few ZIKV insertion mutants replicated, which likely reflects a high degree of functional constraints on the genome. The NS1 gene exhibited distinct mutational tolerances at different stages of the screen. This result may define regions of the NS1 protein that are required for the different stages of the viral life cycle. The ZIKV structural genes showed the highest degree of insertional tolerance. Although the envelope (E) protein exhibited particular flexibility, the highly conserved envelope domain II (EDII) fusion loop of the E protein was intolerant of transposon insertions. The fusion loop is also a target of pan-flavivirus antibodies that are generated against other flaviviruses and neutralize a broad range of dengue virus and ZIKV isolates. The genetic restrictions identified within the epitopes in the EDII fusion loop likely explain the sequence and antigenic conservation of these regions in ZIKV and among multiple flaviviruses. Thus, our results provide insights into the genetic restrictions on ZIKV that may affect the evolution of this virus. IMPORTANCE Zika virus recently emerged as a significant human pathogen. Determining the genetic constraints on Zika virus is important for understanding the factors affecting viral evolution. We used a genome-wide transposon mutagenesis screen to identify where mutations were tolerated in replicating viruses. We found that the genetic regions involved in RNA replication were mostly intolerant of mutations. The genes coding for structural proteins were more permissive to mutations. Despite the flexibility observed in these regions, we found that epitopes bound by broadly reactive antibodies were genetically constrained. This finding may explain

Development of a high-frequency in vivo transposon mutagenesis system for Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942.

PubMed

Watabe, Kazuyuki; Mimuro, Mamoru; Tsuchiya, Tohru

2014-11-01

Synechocystis sp. PCC 6803 (Synechocystis) is the first sequenced photosynthetic organism and has two advantages: natural transformation and light-activated heterotrophic growth. Such characteristics have mainly promoted reverse genetic analysis in this organism, however, to date approximately 50% of genes are still annotated as 'unknown protein' or 'hypothetical protein'. Therefore, forward genetic analysis is required for the identification of significant genes responsible for photosynthesis and other physiological phenomena among the genes of unknown function. The in vivo transposon mutagenesis system is one of the major methods for random mutagenesis. However, present in vivo transposon mutagenesis systems for cyanobacteria face problems such as relatively low frequency of transposition and repeated transposition in the host cells. In this study, we constructed vectors based on a mini-Tn5-derived vector that was designed to prevent repeated transposition. Our vectors carry a hyperactive transposase and optimized recognition sequence of transposase, which were reported to enhance frequency of transposition. Using the vector, we succeeded in highly frequent transposition (9×10(-3) per recipient cell) in Synechocystis. Transposon insertion sites of 10 randomly selected mutants indicated that the insertion sites spread throughout the genome with low sequence dependency. Furthermore, one of the 10 mutants exhibited the slow-growing phenotype, and the mutant was functionally complemented by using our expression vector. Our system also worked with another model cyanobacterium, Synechococcus elongatus PCC 7942, with high frequency. These results indicate that the developed system can be applied to the forward genetic analysis of a broad range of cyanobacteria. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Protein crystallization: Eluding the bottleneck of X-ray crystallography

PubMed Central

Holcomb, Joshua; Spellmon, Nicholas; Zhang, Yingxue; Doughan, Maysaa; Li, Chunying; Yang, Zhe

2017-01-01

To date, X-ray crystallography remains the gold standard for the determination of macromolecular structure and protein substrate interactions. However, the unpredictability of obtaining a protein crystal remains the limiting factor and continues to be the bottleneck in determining protein structures. A vast amount of research has been conducted in order to circumvent this issue with limited success. No single method has proven to guarantee the crystallization of all proteins. However, techniques using antibody fragments, lipids, carrier proteins, and even mutagenesis of crystal contacts have been implemented to increase the odds of obtaining a crystal with adequate diffraction. In addition, we review a new technique using the scaffolding ability of PDZ domains to facilitate nucleation and crystal lattice formation. Although in its infancy, such technology may be a valuable asset and another method in the crystallography toolbox to further the chances of crystallizing problematic proteins. PMID:29051919

Investigation of Trimethyllysine Binding by the HP1 Chromodomain via Unnatural Amino Acid Mutagenesis.

PubMed

Baril, Stefanie A; Koenig, Amber L; Krone, Mackenzie W; Albanese, Katherine I; He, Cyndi Qixin; Lee, Ga Young; Houk, Kendall N; Waters, Marcey L; Brustad, Eric M

2017-12-06

Trimethyllysine (Kme3) reader proteins are targets for inhibition due to their role in mediating gene expression. Although all such reader proteins bind Kme3 in an aromatic cage, the driving force for binding may differ; some readers exhibit evidence for cation-π interactions whereas others do not. We report a general unnatural amino acid mutagenesis approach to quantify the contribution of individual tyrosines to cation binding using the HP1 chromodomain as a model system. We demonstrate that two tyrosines (Y24 and Y48) bind to a Kme3-histone tail peptide via cation-π interactions, but linear free energy trends suggest they do not contribute equally to binding. X-ray structures and computational analysis suggest that the distance and degree of contact between Tyr residues and Kme3 plays an important role in tuning cation-π-mediated Kme3 recognition. Although cation-π interactions have been studied in a number of proteins, this work is the first to utilize direct binding assays, X-ray crystallography, and modeling, to pinpoint factors that influence the magnitude of the individual cation-π interactions.

Induction of mutagenesis and alterations in gene expression by tumorigenic chemicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huberman, E.

1979-01-01

To determine the relationship between mutagenesis and carcinogenesis, a series of eleven polycyclic hydrocarbons with different degrees of carcinogenicity were tested in the cell-mediated mutagenesis assay for the induction of ouabain-resistant mutants. Four carcinogenic hydrocarbons induced ouabain-resistant mutants; five noncarcinogenic hydrocarbons were not mutagenic. Results indicated that there was a relationship between mutagenesis and the degree of carcinogenicity of polycyclic hydrocarbons after enhancement of their metabolism by aminophylline. To study liver carcinogens a system was developed for cocultivating primary liver cells and V79 hamster cells. In this system the nitrosamines and aflatoxins were metabolized by liver cells to intermediates thatmore » were mutagenic to the V79 cells. In experiments using human cells, tumor-promoting phorbol esters induced terminal differentiation while in other studies, in which avian and murine cells were employed, they inhibited differentiation. The results imply that human cells may respond differently from mouse and chicken cells to the biological effects of phorbol diesters. (HLW)« less

Green Fluorescent Protein as a Model for Protein Crystal Growth Studies

NASA Technical Reports Server (NTRS)

Agena, Sabine; Smith, Lori; Karr, Laurel; Pusey, Marc

1998-01-01

Green fluorescent protein (GFP) from jellyfish Aequorea Victoria has become a popular marker for e.g. mutagenesis work. Its fluorescent property, which originates from a chromophore located in the center of the molecule, makes it widely applicable as a research too]. GFP clones have been produced with a variety of spectral properties, such as blue and yellow emitting species. The protein is a single chain of molecular weight 27 kDa and its structure has been determined at 1.9 Angstrom resolution. The combination of GFP's fluorescent property, the knowledge of its several crystallization conditions, and its increasing use in biophysical and biochemical studies, all led us to consider it as a model material for macromolecular crystal growth studies. Initial preparations of GFP were from E.coli with yields of approximately 5 mg/L of culture media. Current yields are now in the 50 - 120 mg/L range, and we hope to further increase this by expression of the GFP gene in the Pichia system. The results of these efforts and of preliminary crystal growth studies will be presented.

Phage transposon mutagenesis.

PubMed

Siegrist, M Sloan; Rubin, Eric J

2009-01-01

Phage transduction is an attractive method of genetic manipulation in mycobacteria. PhiMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an Escherichia coli oriR6 K origin of replication. Mycobacterial transposon mutant libraries produced by PhiMycoMarT7 transduction are amenable to both forward and reverse genetic studies. In this protocol, we detail the preparation of PhiMycoMarT7, including a description of the phage, reconstitution of the phage, purification of plaques, preparation of phage stock, and titering of phage stock. We then describe the transduction procedure and finally outline the isolation of individual transposon mutants.

Increasing protein production rates can decrease the rate at which functional protein is produced

NASA Astrophysics Data System (ADS)

Sharma, Ajeet; O'Brien, Edward

The rate at which soluble, functional protein is produced by the ribosome has recently been found to vary in complex and unexplained ways as various translation-associated rates are altered through synonymous codon substitutions. We combine a well-established ribosome-traffic model with a master-equation model of co-translational domain folding to explore the scenarios that are possible for the protein production rate, J, and the functional-nascent protein production rate, F, as the rates associated with translation are altered. We find that while J monotonically increases as the rates of translation-initiation, -elongation and -termination increase, F can either increase or decrease. F exhibits non-monotonic behavior because increasing these rates can cause a protein to be synthesized more rapidly but provide less time for nascent-protein domains to co-translationally fold thereby producing less functional nascent protein immediately after synthesis. We further demonstrate that these non-monotonic changes in Faffect the post-translational, steady-state levels of functional protein in a similar manner. Our results provide a possible explanation for recent experimental observations that the specific activity of enzymatic proteins can decrease with increased synthesis rates and can in principle be used to rationally-design transcripts to maximize the production of functional nascent protein.

Protein cage assisted metal-protein nanocomposite synthesis: Optimization of loading conditions

NASA Astrophysics Data System (ADS)

Sana, Barindra; Calista, Marcia; Lim, Sierin

2012-11-01

Ferritin is an iron-storage protein in most living systems with a cage-like structure. It has inherent property to form metallic nanocore within its cavity. The metallic core formed within the Archaeoglobus fulgidus ferritin cavity is stabilized by modulating the protein structure by site directed mutagenesis. Encapsulation protocol of various metals within the engineered ferritin cage (AfFtn-AA) is optimized. Dense metallic cores are visualized using electron microscopy and the bound metal was quantified by ICP-spectrometry. The AfFtn-AA is loaded with up to about 350 cobalt, 2000 chromium, and as high as 7000 iron atoms, separately. The metal-protein nanocomposites formed by encapsulation of cobalt, chromium, and iron are studied. Magnetic resonance imaging of the agarose embedded nanocomposites shows brightening of T1-weighted images and signal loss of T2-weighted images with increasing concentration of the nanocomposites. Shortening of magnetic relaxation times in the presence of the nanocomposites confirm their ability to enhance magnetic relaxation rate and suggests that the nanocomposites have potential application as MRI contrast agent.

ENU mutagenesis identifies mice with cardiac fibrosis and hepatic steatosis caused by a mutation in the mitochondrial trifunctional protein beta-subunit.

PubMed

Kao, Hsiao-Jung; Cheng, Ching-Feng; Chen, Yen-Hui; Hung, Shuen-Iu; Huang, Cheng-Chih; Millington, David; Kikuchi, Tateki; Wu, Jer-Yuarn; Chen, Yuan-Tsong

2006-12-15

Using the metabolomics-guided screening coupled to N-ethyl-N-nitrosourea-mediated mutagenesis, we identified mice that exhibited elevated levels of long-chain acylcarnitines. Whole genome homozygosity mapping with 262 SNP markers mapped the disease gene to chromosome 5 where candidate genes Hadha and Hadhb, encoding the mitochondria trifunctional protein (MTP) alpha- and beta-subunits, respectively, are located. Direct sequencing revealed a normal alpha-subunit, but detected a nucleotide T-to-A transversion in exon 14 (c.1210T>A) of beta-subunit (Hadhb) which resulted in a missense mutation of methionine to lysine (M404K). Western blot analysis showed a significant reduction of both the alpha- and beta-subunits, consistent with reduced enzyme activity in both the long-chain 3-hydroxyacyl-CoA dehydrogenase and the long-chain 3-ketoacyl-CoA thiolase activities. These mice had a decreased weight gain and cardiac arrhythmias which manifested from a prolonged PR interval to a complete atrio-ventricular dissociation, and died suddenly between 9 and 16 months of age. Histopathological studies showed multifocal cardiac fibrosis and hepatic steatosis. This mouse model will be useful to further investigate the mechanisms underlying arrhythmogenesis relating to lipotoxic cardiomyopathy and to investigate pathophysiology and treatment strategies for human MTP deficiency.

Increasing the protein content of ice cream.

PubMed

Patel, M R; Baer, R J; Acharya, M R

2006-05-01

Vanilla ice cream was made with a mix composition of 10.5% milk fat, 10.5% milk SNF, 12% beet sugar, and 4% corn syrup solids. None of the batches made contained stabilizer or emulsifier. The control (treatment 1) contained 3.78% protein. Treatments 2 and 5 contained 30% more protein, treatments 3 and 6 contained 60% more protein, and treatments 4 and 7 contained 90% more protein compared with treatment 1 by addition of whey protein concentrate or milk protein concentrate powders, respectively. In all treatments, levels of milk fat, milk SNF, beet sugar, and corn syrup solids were kept constant at 37% total solids. Mix protein content for treatment 1 was 3.78%, treatment 2 was 4.90%, treatment 5 was 4.91%, treatments 3 and 6 were 6.05%, and treatments 4 and 7 were 7.18%. This represented a 29.89, 60.05, 89.95, 29.63, 60.05, and 89.95% increase in protein for treatment 2 through treatment 7 compared with treatment 1, respectively. Milk protein level influenced ice crystal size; with increased protein, the ice crystal size was favorably reduced in treatments 2, 4, and 5 and was similar in treatments 3, 6, and 7 compared with treatment 1. At 1 wk postmanufacture, overall texture acceptance for all treatments was more desirable compared with treatment 1. When evaluating all parameters, treatment 2 with added whey protein concentrate and treatments 5 and 6 with added milk protein concentrate were similar or improved compared with treatment 1. It is possible to produce acceptable ice cream with higher levels of protein.

Alanine Scanning Mutagenesis Identifies an Asparagine–Arginine–Lysine Triad Essential to Assembly of the Shell of the Pdu Microcompartment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sinha, Sharmistha; Cheng, Shouqiang; Sung, Yea Won

2014-06-01

Bacterial microcompartments (MCPs) are the simplest organelles known. They function to enhance metabolic pathways by confining several related enzymes inside an all-protein envelope called the shell. In this study, we investigated the factors that govern MCP assembly by performing scanning mutagenesis on the surface residues of PduA, a major shell protein of the MCP used for 1,2-propanediol degradation. Biochemical, genetic, and structural analysis of 20 mutants allowed us to determine that PduA K26, N29, and R79 are crucial residues that stabilize the shell of the 1,2-propanediol MCP. In addition, we identify two PduA mutants (K37A and K55A) that impair MCPmore » function most likely by altering the permeability of its protein shell. These are the first studies to examine the phenotypic effects of shell protein structural mutations in an MCP system. The findings reported here may be applicable to engineering protein containers with improved stability for biotechnology applications.« less

Characterization and Transposon Mutagenesis of the Maize (Zea mays) Pho1 Gene Family

PubMed Central

Salazar-Vidal, M. Nancy; Acosta-Segovia, Edith; Sánchez-León, Nidia; Ahern, Kevin R.; Brutnell, Thomas P.; Sawers, Ruairidh J. H.

2016-01-01

Phosphorus is an essential nutrient for all plants, but also one of the least mobile, and consequently least available, in the soil. Plants have evolved a series of molecular, metabolic and developmental adaptations to increase the acquisition of phosphorus and to maximize the efficiency of use within the plant. In Arabidopsis (Arabidopsis thaliana), the AtPHO1 protein regulates and facilitates the distribution of phosphorus. To investigate the role of PHO1 proteins in maize (Zea mays), the B73 reference genome was searched for homologous sequences, and four genes identified that were designated ZmPho1;1, ZmPho1;2a, ZmPho1;2b and ZmPho1;3. ZmPho1;2a and ZmPho1;2b are the most similar to AtPHO1, and represent candidate co-orthologs that we hypothesize to have been retained following whole genome duplication. Evidence was obtained for the production of natural anti-sense transcripts associated with both ZmPho1;2a and ZmPho1;2b, suggesting the possibility of regulatory crosstalk between paralogs. To characterize functional divergence between ZmPho1;2a and ZmPho1;2b, a program of transposon mutagenesis was initiated using the Ac/Ds system, and, here, we report the generation of novel alleles of ZmPho1;2a and ZmPho1;2b. PMID:27648940

Scaffold diversification enhances effectiveness of a superlibrary of hyperthermophilic proteins.

PubMed

Hussain, Mahmud; Gera, Nimish; Hill, Andrew B; Rao, Balaji M

2013-01-18

The use of binding proteins from non-immunoglobulin scaffolds has become increasingly common in biotechnology and medicine. Typically, binders are isolated from a combinatorial library generated by mutating a single scaffold protein. In contrast, here we generated a "superlibrary" or "library-of-libraries" of 4 × 10(8) protein variants by mutagenesis of seven different hyperthermophilic proteins; six of the seven proteins have not been used as scaffolds prior to this study. Binding proteins for five different model targets were successfully isolated from this library. Binders obtained were derived from five out of the seven scaffolds. Strikingly, binders from this modestly sized superlibrary have affinities comparable or higher than those obtained from a library with 1000-fold higher sequence diversity but derived from a single stable scaffold. Thus scaffold diversification, i.e., randomization of multiple different scaffolds, is a powerful alternate strategy for combinatorial library construction.

Mutational analysis of Kaposica reveals that bridging of MG2 and CUB domains of target protein is crucial for the cofactor activity of RCA proteins

PubMed Central

Gautam, Avneesh Kumar; Panse, Yogesh; Ghosh, Payel; Reza, Malik Johid; Mullick, Jayati; Sahu, Arvind

2015-01-01

The complement system has evolved to annul pathogens, but its improper regulation is linked with diseases. Efficient regulation of the system is primarily provided by a family of proteins termed regulators of complement activation (RCA). The knowledge of precise structural determinants of RCA proteins critical for imparting the regulatory activities and the molecular events underlying the regulatory processes, nonetheless, is still limited. Here, we have dissected the structural requirements of RCA proteins that are crucial for one of their two regulatory activities, the cofactor activity (CFA), by using the Kaposi’s sarcoma-associated herpesvirus RCA homolog Kaposica as a model protein. We have scanned the entire Kaposica molecule by sequential mutagenesis using swapping and site-directed mutagenesis, which identified residues critical for its interaction with C3b and factor I. Mapping of these residues onto the modeled structure of C3b–Kaposica–factor I complex supported the mutagenesis data. Furthermore, the model suggested that the C3b-interacting residues bridge the CUB (complement C1r-C1s, Uegf, Bmp1) and MG2 (macroglobulin-2) domains of C3b. Thus, it seems that stabilization of the CUB domain with respect to the core of the C3b molecule is central for its CFA. Identification of CFA-critical regions in Kaposica guided experiments in which the equivalent regions of membrane cofactor protein were swapped into decay-accelerating factor. This strategy allowed CFA to be introduced into decay-accelerating factor, suggesting that viral and human regulators use a common mechanism for CFA. PMID:26420870

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pepinsky, R. Blake; Silvian, Laura; Berkowitz, Steven A.

2010-11-15

Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti-LINGO-1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for itsmore » 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.« less

Modulating the activity of protein conjugated to gold nanoparticles by site-directed orientation and surface density of bound protein.

PubMed

Liu, Feng; Wang, Lei; Wang, Hongwei; Yuan, Lin; Li, Jingwen; Brash, John Law; Chen, Hong

2015-02-18

The key property of protein-nanoparticle conjugates is the bioactivity of the protein. The ability to accurately modulate the activity of protein on the nanoparticles at the interfaces is important in many applications. In the work reported here, modulation of the activity of protein-gold nanoparticle (AuNP) conjugates by specifically orienting the protein and by varying the surface density of the protein was investigated. Different orientations were achieved by introducing cysteine (Cys) residues at specific sites for binding to gold. We chose Escherichia coli inorganic pyrophosphatase (PPase) as a model protein and used site-directed mutagenesis to generate two mutant types (MTs) with a single Cys residue on the surface: MT1 with Cys near the active center and MT2 with Cys far from the active center. The relative activities of AuNP conjugates with wild type (WT), MT1, and MT2 were found to be 44.8%, 68.8%, and 91.2% of native PPase in aqueous solution. Site-directed orientation with the binding site far from the active center thus allowed almost complete preservation of the protein activity. The relative activity of WT and MT2 conjugates did not change with the surface density of the protein, while that of MT1 increased significantly with increasing surface density. These results demonstrate that site-directed orientation and surface density can both modulate the activity of proteins conjugated to AuNP and that orientation has a greater effect than density. Furthermore, increasing the surface density of the specifically oriented protein MT2, while having no significant effect on the specific activity of the protein, still allowed increased protein loading on the AuNP and thus increased the total protein activity. This is of great importance in the study on the interface of protein and nanoparticle and the applications for enzyme immobilization, drug delivery, and biocatalysis.

β-lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity

PubMed Central

Gutierrez, A.; Laureti, L.; Crussard, S.; Abida, H.; Rodríguez-Rojas, A.; Blázquez, J.; Baharoglu, Z.; Mazel, D.; Darfeuille, F.; Vogel, J.; Matic, I.

2013-01-01

Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of β-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of β-lactam antibiotics induce the RpoS regulon. RpoS-regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies. PMID:23511474

Combined mutagenesis of Rhodosporidium toruloides for improved production of carotenoids and lipids.

PubMed

Zhang, Chaolei; Shen, Hongwei; Zhang, Xibin; Yu, Xue; Wang, Han; Xiao, Shan; Wang, Jihui; Zhao, Zongbao K

2016-10-01

To improve production of lipids and carotenoids by the oleaginous yeast Rhodosporidium toruloides by screening mutant strains. Upon physical mutagenesis of the haploid strain R. toruloides np11 with an atmospheric and room temperature plasma method followed by chemical mutagenesis with nitrosoguanidine, a mutant strain, R. toruloides XR-2, formed dark-red colonies on a screening plate. When cultivated in nitrogen-limited media, XR-2 cells grew slower but accumulated 0.23 g lipids/g cell dry wt and 0.75 mg carotenoids/g CDW. To improve its production capacity, different amino acids and vitamins were supplemented. p-Aminobenzoic acid and tryptophan had beneficial effects on cell growth. When cultivated in nitrogen-limited media in the presence of selected vitamins, XR-2 accumulated 0.41 g lipids/g CDW and 0.69 mg carotenoids/g CDW. A mutant R. toruloides strain with improved production profiles for lipids and carotenoids was obtained, indicating its potential to use combined mutagenesis for a more productive phenotype.

Insertional mutagenesis using Tnt1 retrotransposon in potato

USDA-ARS?s Scientific Manuscript database

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

Methods for targetted mutagenesis in gram-positive bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Yunfeng

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.

Natural mutagenesis of human genomes by endogenous retrotransposons.

PubMed

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

2010-06-25

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

Endotoxin increases pulmonary vascular protein permeability in the dog

DOE Office of Scientific and Technical Information (OSTI.GOV)

Welsh, C.H.; Dauber, I.M.; Weil, J.V.

Endotoxin increases pulmonary vascular permeability consistently in some species but fails to reliably cause injury in the dog. We wondered whether this phenomenon depended on the method of injury assessment, as others have relied on edema measurement; we quantified injury by monitoring the rate of extravascular protein accumulation. /sup 113m/In-labeled protein and /sup 99m/Tc-labeled erythrocytes were injected into anesthetized dogs and monitored by an externally placed lung probe. A protein leak index, the rate of extravascular protein accumulation, was derived from the rate of increase in lung protein counts corrected for changes in intravascular protein activity. After administration of Salmonellamore » enteriditis endotoxin (4 micrograms/kg), the protein leak index was elevated 2.5-fold (41.1 +/- 4.6 X 10(-4) min-1) compared with control (16.0 +/- 2.8 X 10(-4) min-1). In contrast, wet-to-dry weight ratios failed to increase after endotoxin (4.6 +/- 0.8 vs. control values of 4.2 +/- 0.5 g/g dry bloodless lung). However, we observed that endotoxin increased lung dry weight (per unit body weight), which may have attenuated the change in wet-to-dry weight ratios. To determine whether low microvascular pressures following endotoxin attenuated edema formation, we increased pulmonary arterial wedge pressures in five dogs by saline infusion, which caused an increase in wet-to-dry weight ratios following endotoxin but no change in the five controls. We conclude that low dose endotoxin causes pulmonary vascular protein leak in the dog while edema formation is minimal or absent.« less

Targeted mutagenesis using CRISPR/Cas in inbred potatoes

USDA-ARS?s Scientific Manuscript database

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...

Generation of Peanut Drought Tolerant Plants by Pingyangmycin-Mediated In Vitro Mutagenesis and Hydroxyproline-Resistance Screening

PubMed Central

Qiao, Lixian; Sun, Shimeng; Hu, Xiaohui; Chen, Jing; Wang, Jingshan

2015-01-01

In order to enlarge the potential resources of drought-tolerant peanuts, we conducted in vitro mutagenesis with Pingyangmycin (PYM) as the mutagen as well as directed screening on a medium supplemented with Hydroxyproline (HYP). After being extracted from mature seeds (cv. Huayu 20), the embryonic leaflets were cultured on somatic embryogenesis-induction medium with 4 mg/L PYM and the generated embryos were successively transferred to a germination medium with 4 and then 8 mmol/L HYP to screen HYP-tolerant plantlets. After that, these plantlets were grafted and transplanted to the experimental field. In the next generation, all seeds were sown in the field, and phenotype variation and trait segregation can be observed in most of the offspring (M2 generation). The M3 generation individuals were subjected to drought stress at the seedling stages. The activities of SOD and POD were substantially increased in eight offspring of 11 HYP-tolerant, regenerated plants than in their mutagenic parents. To determine the correlation between mutant phenotypes and genomic modification, we carried out a comparison of the DNA polymorphisms between the mutagenic parents and 13 M3 generation individuals from different HYP-tolerant, regenerated plants with SSR primers. Results showed that most mutants and parent plants had signs of polymorphisms. Under drought stress, some M3 generation individuals of 10 original HYP-tolerant, regenerated plants produced more pods than the mutagenic parent; twenty individuals among them produced >60 g pods/plant. M4-generation seeds were tested for quality characteristics by Near Infrared Spectroscopy (NIS) and nine individuals with higher protein content (>30%) and 21 individuals with higher oil content (>58%) were screened. We concluded that the use of PYM-based in vitro mutagenesis in combination with directed screening with HYP is effective for the creation of potential drought-tolerant mutants of peanut. PMID:25826431

Directed molecular evolution to design advanced red fluorescent proteins.

PubMed

Subach, Fedor V; Piatkevich, Kiryl D; Verkhusha, Vladislav V

2011-11-29

Fluorescent proteins have become indispensable imaging tools for biomedical research. Continuing progress in fluorescence imaging, however, requires probes with additional colors and properties optimized for emerging techniques. Here we summarize strategies for development of red-shifted fluorescent proteins. We discuss possibilities for knowledge-based rational design based on the photochemistry of fluorescent proteins and the position of the chromophore in protein structure. We consider advances in library design by mutagenesis, protein expression systems and instrumentation for high-throughput screening that should yield improved fluorescent proteins for advanced imaging applications.

Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis.

PubMed

Castro-Cerritos, Karla Viridiana; Yasbin, Ronald E; Robleto, Eduardo A; Pedraza-Reyes, Mario

2017-02-15

The Gram-positive microorganism Bacillus subtilis relies on a single class Ib ribonucleotide reductase (RNR) to generate 2'-deoxyribonucleotides (dNDPs) for DNA replication and repair. In this work, we investigated the influence of RNR levels on B. subtilis stationary-phase-associated mutagenesis (SPM). Since RNR is essential in this bacterium, we engineered a conditional mutant of strain B. subtilis YB955 (hisC952 metB5 leu427) in which expression of the nrdEF operon was modulated by isopropyl-β-d-thiogalactopyranoside (IPTG). Moreover, genetic inactivation of ytcG, predicted to encode a repressor (NrdR) of nrdEF in this strain, dramatically increased the expression levels of a transcriptional nrdE-lacZ fusion. The frequencies of mutations conferring amino acid prototrophy in three genes were measured in cultures under conditions that repressed or induced RNR-encoding genes. The results revealed that RNR was necessary for SPM and overexpression of nrdEF promoted growth-dependent mutagenesis and SPM. We also found that nrdEF expression was induced by H 2 O 2 and such induction was dependent on the master regulator PerR. These observations strongly suggest that the metabolic conditions operating in starved B. subtilis cells increase the levels of RNR, which have a direct impact on SPM. Results presented in this study support the concept that the adverse metabolic conditions prevailing in nutritionally stressed bacteria activate an oxidative stress response that disturbs ribonucleotide reductase (RNR) levels. Such an alteration of RNR levels promotes mutagenic events that allow Bacillus subtilis to escape from growth-limited conditions. Copyright © 2017 American Society for Microbiology.

DC-Analyzer-facilitated combinatorial strategy for rapid directed evolution of functional enzymes with multiple mutagenesis sites.

PubMed

Wang, Xiong; Zheng, Kai; Zheng, Huayu; Nie, Hongli; Yang, Zujun; Tang, Lixia

2014-12-20

Iterative saturation mutagenesis (ISM) has been shown to be a powerful method for directed evolution. In this study, the approach was modified (termed M-ISM) by combining the single-site saturation mutagenesis method with a DC-Analyzer-facilitated combinatorial strategy, aiming to evolve novel biocatalysts efficiently in the case where multiple sites are targeted simultaneously. Initially, all target sites were explored individually by constructing single-site saturation mutagenesis libraries. Next, the top two to four variants in each library were selected and combined using the DC-Analyzer-facilitated combinatorial strategy. In addition to site-saturation mutagenesis, iterative saturation mutagenesis also needed to be performed. The advantages of M-ISM over ISM were that the screening effort is greatly reduced, and the entire M-ISM procedure was less time-consuming. The M-ISM strategy was successfully applied to the randomization of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) when five interesting sites were targeted simultaneously. After screening 900 clones in total, six positive mutants were obtained. These mutants exhibited 4.0- to 9.3-fold higher k(cat) values than did the wild-type HheC toward 1,3-dichloro-2-propanol. However, with the ISM strategy, the best hit showed a 5.9-fold higher k(cat) value toward 1,3-DCP than the wild-type HheC, which was obtained after screening 4000 clones from four rounds of mutagenesis. Therefore, M-ISM could serve as a simple and efficient version of ISM for the randomization of target genes with multiple positions of interest.

[Corpuscular mutagenesis and its prevention].

PubMed

Daugel'-Dauge, N O; Durnev, A D; Kulakova, A V; Seredenin, S B; Velichkovskiĭ, B T

1995-01-01

The carcinogenic and mutagenic activity of dust containing chrysotile-asbestos and zeolites, as well as the role of active oxygen species in their cytotoxic and mutagenic actions are discussed. Superoxide dismutase (50 mg/ml) was demonstrated to prevent the mutagenic effects of chrysotile-asbestos and latex, catalase (20 mg/ml) to prevent the same of zeolites in experiments on cultured human whole blood. The intraperitoneal administration of dusts of chrysotile-asbestos and zeolites in a dose of 50 mg/kg to C57B1/6 mice was found to elevate the count of cells with chromosomal aberrations in the peritoneal liquid and bone marrow cells of mice, which was dependent on dust exposure time. It was revealed that ascorbic acid, rutin, chemically modified flavonoid of Scutellaria Baicalensis Georgy, drugs such as bemitil and thomersol in the broad range of concentrations (10(-7)-10(-3) M) decreased or completely reduced the clustogenic action of zeolites and chrysotile-asbestos on cultured human whole blood. The ability of bemitil (1.8-19 mg/kg) rather than the others to prevent the mutagenic effect of chrysotile-asbestos was confirmed by the method of recording chromosomal aberrations in the cells of peritoneal liquid and bone marrow in mice. The findings suggest that the mutagenic effects of the corpuscular xenobiotics under study are mediated by active oxygen species and that the use of the models in vitro and in vivo is adequate for investigations into corpuscular mutagenesis. Based on their own data and literature data, the authors have defined possible lines of further research of corpuscular mutagenesis.

Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.

PubMed

Reid-Bayliss, Kate S; Loeb, Lawrence A

2017-08-29

Transcriptional mutagenesis (TM) due to misincorporation during RNA transcription can result in mutant RNAs, or epimutations, that generate proteins with altered properties. TM has long been hypothesized to play a role in aging, cancer, and viral and bacterial evolution. However, inadequate methodologies have limited progress in elucidating a causal association. We present a high-throughput, highly accurate RNA sequencing method to measure epimutations with single-molecule sensitivity. Accurate RNA consensus sequencing (ARC-seq) uniquely combines RNA barcoding and generation of multiple cDNA copies per RNA molecule to eliminate errors introduced during cDNA synthesis, PCR, and sequencing. The stringency of ARC-seq can be scaled to accommodate the quality of input RNAs. We apply ARC-seq to directly assess transcriptome-wide epimutations resulting from RNA polymerase mutants and oxidative stress.

Applications of CRISPR/Cas9 technology for targeted mutagenesis, gene replacement and stacking of genes in higher plants.

PubMed

Luo, Ming; Gilbert, Brian; Ayliffe, Michael

2016-07-01

Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described.

Rational and random mutagenesis of firefly luciferase to identify an efficient emitter of red bioluminescence

NASA Astrophysics Data System (ADS)

Branchini, Bruce R.; Southworth, Tara L.; Khattak, Neelum F.; Murtiashaw, Martha H.; Fleet, Sarah E.

2004-06-01

Firefly luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a bioluminescence reporter of gene expression. One particularly exciting application is in the area of in vivo bioluminescence imaging. Our interest is in developing improved reagents by identifying Photinus pyralis luciferase mutants that efficiently emit red bioluminescence. In this way, the proven advantages of the P. pyralis protein can be combined with the potential advantages of a red-shifted emitter. Using site-directed mutagenesis techniques, we have identified many mutants emitting red bioluminescence. Unfortunately, these enzymes generally have significantly decreased bioluminescence activity. Interestingly, we discovered a mutation, Ile351Ala, that produced a moderate 16 nm red-shift, while maintaining excellent bioluminescence activity. We then undertook a random mutagenesis approach to identify luciferase mutants that emit further red-shifted bioluminescence with minimal loss of activity. Libraries of mutants were created using an error-prone PCR method and the Ile351Ala luciferase mutant as the template DNA. The libraries were screened by in vivo bacterial assays and the promising mutants were purified to enable accurate determination of bioluminescence emission spectra and total bioluminescence activity. We will report the characterization results, including the identification of the randomly altered amino acids, of several mutants that catalyze bioluminescence with emission maxima of approximately 600 nm.

Improvement of Lead Tolerance of Saccharomyces cerevisiae by Random Mutagenesis of Transcription Regulator SPT3.

PubMed

Zhu, Liying; Gao, Shan; Zhang, Hongman; Huang, He; Jiang, Ling

2018-01-01

Bioremediation of heavy metal pollution with biomaterials such as bacteria and fungi usually suffer from limitations because of microbial sensitivity to high concentration of heavy metals. Herein, we adopted a novel random mutagenesis technique called RAISE to manipulate the transcription regulator SPT3 of Saccharomyces cerevisiae to improve cell lead tolerance. The best strain Mutant VI was selected from the random mutagenesis libraries on account of the growth performance, with higher specific growth rate than the control strain (0.068 vs. 0.040 h -1 ) at lead concentration as high as 1.8 g/L. Combined with the transcriptome analysis of S. cerevisiae, expressing the SPT3 protein was performed to make better sense of the global regulatory effects of SPT3. The data analysis revealed that 57 of S. cerevisiae genes were induced and 113 genes were suppressed, ranging from those for trehalose synthesis, carbon metabolism, and nucleotide synthesis to lead resistance. Especially, the accumulation of intracellular trehalose in S. cerevisiae under certain conditions of stress is considered important to lead resistance. The above results represented that SPT3 was acted as global transcription regulator in the exponential phase of strain and accordingly improved heavy metal tolerance in the heterologous host S. cerevisiae. The present study provides a route to complex phenotypes that are not readily accessible by traditional methods.

Mapping hydration dynamics and coupled water-protein fluctuations around a protein surface

NASA Astrophysics Data System (ADS)

Zhang, Luyuan; Wang, Lijuan; Kao, Ya-Ting; Qiu, Weihong; Yang, Yi; Okobiah, Oghaghare; Zhong, Dongping

2009-03-01

Elucidation of the molecular mechanism of water-protein interactions is critical to understanding many fundamental aspects of protein science, such as protein folding and misfolding and enzyme catalysis. We recently carried out a global mapping of protein-surface hydration dynamics around a globular α-helical protein apomyoglobin. The intrinsic optical probe tryptophan was employed to scan the protein surface one at a time by site-specific mutagenesis. With femtosecond resolution, we mapped out the dynamics of water-protein interactions with more than 20 mutants and for two states, native and molten globular. A robust bimodal distribution of time scales was observed, representing two types of water motions: local relaxation and protein-coupled fluctuations. The time scales show a strong correlation with the local protein structural rigidity and chemical identity. We also resolved two distinct contributions to the overall Stokes-shifts from the two time scales. These results are significant to understanding the role of hydration water on protein structural stability, dynamics and function.

Intermolecular cleavage by UmuD-like mutagenesis proteins

PubMed Central

McDonald, John P.; Frank, Ekaterina G.; Levine, Arthur S.; Woodgate, Roger

1998-01-01

The activity of a number of proteins is regulated by self-processing reactions. Elegant examples are the cleavage of the prokaryotic LexA and λCI transcriptional repressors and the UmuD-like mutagenesis proteins. Various studies support the hypothesis that LexA and λCI cleavage reactions are predominantly intramolecular in nature. The recently described crystal structure of the Escherichia coli UmuD′ protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 Å away from the catalytic active site. We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and λCI, occurs via an intermolecular rather than intramolecular reaction. To test this hypothesis, we introduced into E. coli compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins. Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature. Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable E. coli strains. Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed in vitro in the presence of the wild-type UmuD′ protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD′) can also act as a classical enzyme. PMID:9465040

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

PubMed

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

2016-07-31

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

Structure of an Engineered β-Lactamase Maltose Binding Protein Fusion Protein: Insights into Heterotropic Allosteric Regulation

PubMed Central

Ke, Wei; Laurent, Abigail H.; Armstrong, Morgan D.; Chen, Yuchao; Smith, William E.; Liang, Jing; Wright, Chapman M.; Ostermeier, Marc; van den Akker, Focco

2012-01-01

Engineering novel allostery into existing proteins is a challenging endeavor to obtain novel sensors, therapeutic proteins, or modulate metabolic and cellular processes. The RG13 protein achieves such allostery by inserting a circularly permuted TEM-1 β-lactamase gene into the maltose binding protein (MBP). RG13 is positively regulated by maltose yet is, serendipitously, inhibited by Zn2+ at low µM concentration. To probe the structure and allostery of RG13, we crystallized RG13 in the presence of mM Zn2+ concentration and determined its structure. The structure reveals that the MBP and TEM-1 domains are in close proximity connected via two linkers and a zinc ion bridging both domains. By bridging both TEM-1 and MBP, Zn2+ acts to “twist tie” the linkers thereby partially dislodging a linker between the two domains from its original catalytically productive position in TEM-1. This linker 1 contains residues normally part of the TEM-1 active site including the critical β3 and β4 strands important for activity. Mutagenesis of residues comprising the crystallographically observed Zn2+ site only slightly affected Zn2+ inhibition 2- to 4-fold. Combined with previous mutagenesis results we therefore hypothesize the presence of two or more inter-domain mutually exclusive inhibitory Zn2+ sites. Mutagenesis and molecular modeling of an intact TEM-1 domain near MBP within the RG13 framework indicated a close surface proximity of the two domains with maltose switching being critically dependent on MBP linker anchoring residues and linker length. Structural analysis indicated that the linker attachment sites on MBP are at a site that, upon maltose binding, harbors both the largest local Cα distance changes and displays surface curvature changes, from concave to relatively flat becoming thus less sterically intrusive. Maltose activation and zinc inhibition of RG13 are hypothesized to have opposite effects on productive relaxation of the TEM-1 β3 linker region via

Protein and Antibody Engineering by Phage Display.

PubMed

Frei, J C; Lai, J R

2016-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. © 2016 Elsevier Inc. All rights reserved.

Protein and Antibody Engineering by Phage Display

PubMed Central

Frei, J.C.; Lai, J.R.

2017-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. PMID:27586328

Regulation of MVM NS1 by protein kinase C: impact of mutagenesis at consensus phosphorylation sites on replicative functions and cytopathic effects.

PubMed

Corbau, R; Duverger, V; Rommelaere, J; Nüesch, J P

2000-12-05

Minute virus of mice NS1, an 83-kDa mainly nuclear phosphoprotein, is the only viral nonstructural protein required in all cell types and it is involved in multiple processes necessary for virus propagation. The diversity of functions assigned to NS1, together with the variation of its complex phosphorylation pattern during infection, suggested that the various activities of NS1 could be regulated by distinct phosphorylation events. So far, it has been demonstrated that NS1 replicative functions, in particular, DNA-unwinding activities, are regulated by protein kinase C (PKC), as exemplified by the modulation of NS1 helicase activity by PKClambda phosphorylation. In order to determine further impact of phosphorylation on NS1 functions, including the induction of cytopathic effects, a mutational approach was pursued in order to produce NS1 variants harboring amino acid substitutions at candidate PKC target residues. Besides the determination of two additional in vivo phosphorylation sites in NS1, this mutagenesis allowed the segregation of distinct NS1 functions from one another, generating NS1 variants with a distinct activity profile. Thus, we obtained NS1 mutants that were fully proficient for trans activation of the viral P38 promoter, while being impaired in their replicative functions. Moreover, the alterations of specific PKC phosphorylation sites gave rise to NS1 polypeptides that exerted reduced cytotoxicity, leading to sustained gene expression, while keeping functions necessary for progeny virus production, i.e., viral DNA replication and activation of the capsid gene promoter. These data suggested that in the course of a viral infection, NS1 may undergo a shift from productive to cytotoxic functions as a result of a phosphorylation-dependent regulation. Copyright 2000 Academic Press.

Significantly enhancing recombinant alkaline amylase production in Bacillus subtilis by integration of a novel mutagenesis-screening strategy with systems-level fermentation optimization.

PubMed

Ma, Yingfang; Shen, Wei; Chen, Xianzhong; Liu, Long; Zhou, Zhemin; Xu, Fei; Yang, Haiquan

2016-01-01

Alkaline amylase has significant potential for applications in the textile, paper and detergent industries, however, low yield of which cannot meet the requirement of industrial application. In this work, a novel ARTP mutagenesis-screening method and fermentation optimization strategies were used to significantly improve the expression level of recombinant alkaline amylase in B. subtilis 168. The activity of alkaline amylase in mutant B. subtilis 168 mut-16# strain was 1.34-fold greater than that in the wild-type, and the highest specific production rate was improved from 1.31 U/(mg·h) in the wild-type strain to 1.57 U/(mg·h) in the mutant strain. Meanwhile, the growth of B. subtilis was significantly enhanced by ARTP mutagenesis. When the agitation speed was 550 rpm, the highest activity of recombinant alkaline amylase was 1.16- and 1.25-fold of the activities at 450 and 650 rpm, respectively. When the concentration of soluble starch and soy peptone in the initial fermentation medium was doubled, alkaline amylase activity was increased 1.29-fold. Feeding hydrolyzed starch and soy peptone mixture or glucose significantly improved cell growth, but inhibited the alkaline amylase production in B. subtilis 168 mut-16#. The highest alkaline amylase activity by feeding hydrolyzed starch reached 591.4 U/mL, which was 1.51-fold the activity by feeding hydrolyzed starch and soy peptone mixture. Single pulse feeding-based batch feeding at 10 h favored the production of alkaline amylase in B. subtilis 168 mut-16#. The results indicated that this novel ARTP mutagenesis-screening method could significantly improve the yield of recombinant proteins in B. subtilis . Meanwhile, fermentation optimization strategies efficiently promoted expression of recombinant alkaline amylase in B. subtilis 168 mut-16#. These findings have great potential for facilitating the industrial-scale production of alkaline amylase and other enzymes, using B. subtilis cultures as microbial cell

Isolating Escherichia coli strains for recombinant protein production.

PubMed

Schlegel, Susan; Genevaux, Pierre; de Gier, Jan-Willem

2017-03-01

Escherichia coli has been widely used for the production of recombinant proteins. To improve protein production yields in E. coli, directed engineering approaches have been commonly used. However, there are only few reported examples of the isolation of E. coli protein production strains using evolutionary approaches. Here, we first give an introduction to bacterial evolution and mutagenesis to set the stage for discussing how so far selection- and screening-based approaches have been used to isolate E. coli protein production strains. Finally, we discuss how evolutionary approaches may be used in the future to isolate E. coli strains with improved protein production characteristics.

Structural basis of a rationally rewired protein-protein interface critical to bacterial signaling

PubMed Central

Podgornaia, Anna I.; Casino, Patricia; Marina, Alberto; Laub, Michael T.

2013-01-01

Summary Two-component signal transduction systems typically involve a sensor histidine kinase that specifically phosphorylates a single, cognate response regulator. This protein-protein interaction relies on molecular recognition via a small set of residues in each protein. To better understand how these residues determine the specificity of kinase-substrate interactions, we rationally rewired the interaction interface of a Thermotoga maritima two-component system, HK853-RR468, to match that found in a different two-component system, E. coli PhoR-PhoB. The rewired proteins interacted robustly with each other, but no longer interacted with the parent proteins. Analysis of the crystal structures of the wild-type and mutant protein complexes, along with a systematic mutagenesis study, reveals how individual mutations contribute to the rewiring of interaction specificity. Our approach and conclusions have implications for studies of other protein-protein interactions, protein evolution, and the design of novel protein interfaces. PMID:23954504

Photoswitchable red fluorescent protein with a large Stokes shift

PubMed Central

Piatkevich, Kiryl D.; English, Brian P.; Malashkevich, Vladimir N.; Xiao, Hui; Almo, Steven C.; Singer, Robert H.; Verkhusha, Vladislav V.

2014-01-01

SUMMARY Subclass of fluorescent proteins, large Stokes shift fluorescent proteins, is characterized by their increased spread between the excitation and emission maxima. Here we report a photoswitchable variant of a red fluorescent protein with a large Stokes shift, PSLSSmKate, which initially exhibits excitation/emission at 445/622 nm, but irradiation with violet light photoswitches PSLSSmKate into a common red form with excitation/emission at 573/621 nm. We characterize spectral, photophysical and biochemical properties of PSLSSmKate in vitro and in mammalian cells, and determine its crystal structure in the large Stokes shift form. Mass-spectrometry, mutagenesis and spectroscopic analysis of PSLSSmKate allow us to propose molecular mechanisms for the large Stokes shift, pH dependence and light-induced chromophore transformation. We demonstrate applicability of PSLSSmKate to superresolution PALM microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects. PMID:25242289

Fluorometric method of quantitative cell mutagenesis

DOEpatents

Dolbeare, Frank A.

1982-01-01

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

Fluorometric method of quantitative cell mutagenesis

DOEpatents

Dolbeare, F.A.

1980-12-12

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

Environmental mutagenesis during the end-Permian ecological crisis

PubMed Central

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

2004-01-01

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

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

PubMed Central

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

2009-01-01

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

Protein kinase A-dependent increase in WAVE2 expression induced by the focal adhesion protein vinexin.

PubMed

Mitsushima, Masaru; Sezaki, Takuhito; Akahane, Rie; Ueda, Kazumitsu; Suetsugu, Shiro; Takenawa, Tadaomi; Kioka, Noriyuki

2006-03-01

The focal adhesion protein vinexin is a member of a family of adaptor proteins that are thought to participate in the regulation of cell adhesion, cytoskeletal reorganization, and growth factor signaling. Here, we show that vinexin beta increases the amount of and reduces the mobility on SDS-PAGE of Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) 2 protein, which is a key factor modulating actin polymerization in migrating cells. This mobility retardation disappeared after in vitro phosphatase treatment. Co-immunoprecipitation assays revealed the interaction of vinexin beta with WAVE2 as well as WAVE1 and N-WASP. Vinexin beta interacts with the proline-rich region of WAVE2 through the first and second SH3 domains of vinexin beta. Mutations disrupting the interaction impaired the ability of vinexin beta to increase the amount of WAVE2 protein. Treatments with proteasome inhibitors increased the amount of WAVE2, but did not have an additive effect with vinexin beta. Inhibition of protein kinase A (PKA) activity suppressed the vinexin-induced increase in WAVE2 protein, while activation of PKA increased WAVE2 expression without vinexin beta. These results suggest that vinexin beta regulates the proteasome-dependent degradation of WAVE2 in a PKA-dependent manner.

Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice.

PubMed

Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

2015-10-01

Frequency of CRISPR/Cas9-mediated targeted mutagenesis varies depending on Cas9 expression level and culture period of rice callus. Recent reports have demonstrated that the CRISPR/Cas9 system can function as a sequence-specific nuclease in various plant species. Induction of mutation in proliferating tissue during embryogenesis or in germline cells is a practical means of generating heritable mutations. In the case of plant species in which cultured cells are used for transformation, non-chimeric plants can be obtained when regeneration occurs from mutated cells. Since plantlets are regenerated from both mutated and non-mutated cells in a random manner, any increment in the proportion of mutated cells in Cas9- and guide RNA (gRNA)-expressing cells will help increase the number of plants containing heritable mutations. In this study, we examined factors affecting mutation frequency in rice calli. Following sequential transformation of rice calli with Cas9- and gRNA- expression constructs, the mutation frequency in independent Cas9 transgenic lines was analyzed. A positive correlation between Cas9 expression level and mutation frequency was found. This positive relationship was observed regardless of whether the transgene or an endogenous gene was used as the target for CRISPR/Cas9-mediated mutagenesis. Furthermore, we found that extending the culture period increased the proportion of mutated cells as well as the variety of mutations obtained. Because mutated and non-mutated cells might proliferate equally, these results suggest that a prolonged tissue culture period increases the chance of inducing de novo mutations in non-mutated cells. This fundamental knowledge will help improve systems for obtaining non-chimeric regenerated plants in many plant species.

Chronic low-dose ultraviolet-induced mutagenesis in nucleotide excision repair-deficient cells.

PubMed

Haruta, Nami; Kubota, Yoshino; Hishida, Takashi

2012-09-01

UV radiation induces two major types of DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidine photoproducts, which are both primarily repaired by nucleotide excision repair (NER). Here, we investigated how chronic low-dose UV (CLUV)-induced mutagenesis occurs in rad14Δ NER-deficient yeast cells, which lack the yeast orthologue of human xeroderma pigmentosum A (XPA). The results show that rad14Δ cells have a marked increase in CLUV-induced mutations, most of which are C→T transitions in the template strand for transcription. Unexpectedly, many of the CLUV-induced C→T mutations in rad14Δ cells are dependent on translesion synthesis (TLS) DNA polymerase η, encoded by RAD30, despite its previously established role in error-free TLS. Furthermore, we demonstrate that deamination of cytosine-containing CPDs contributes to CLUV-induced mutagenesis. Taken together, these results uncover a novel role for Polη in the induction of C→T transitions through deamination of cytosine-containing CPDs in CLUV-exposed NER deficient cells. More generally, our data suggest that Polη can act as both an error-free and a mutagenic DNA polymerase, depending on whether the NER pathway is available to efficiently repair damaged templates.

Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

NASA Technical Reports Server (NTRS)

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

1999-01-01

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

Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

NASA Technical Reports Server (NTRS)

Crawford, Lisa; Karr, Laurel; Pusey, Marc

1998-01-01

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

CRISPR/Cas9-mediated targeted mutagenesis in grape

PubMed Central

Ban, Yusuke; Azuma, Akifumi; Onoue, Noriyuki; Moriguchi, Takaya; Yamamoto, Toshiya; Toki, Seiichi

2017-01-01

RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape—an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased. PMID:28542349

Cancer gene discovery: exploiting insertional mutagenesis

PubMed Central

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

2013-01-01

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

Catalytic site of human protein-glucosylgalactosylhydroxylysine glucosidase: Three crucial carboxyl residues were determined by cloning and site-directed mutagenesis.

PubMed

Hamazaki, Hideaki; Hamazaki, Michiko Horikawa

2016-01-15

Protein-glucosylgalactosylhydroxylysine glucosidase (PGGHG; EC3.2.1.107) cleaves glucose from disaccharide unit (Glc-α1,2-Gal) linked to hydroxylysine residues of collagen. In the present paper we first show that PGGHG is the product of ATHL1 gene as follows. (1) PGGHG was purified from chick embryos and digested with trypsin. LC-MS/MS analysis suggested the tryptic-peptides were from the ATHL1 gene product. (2) Chick embryo ATHL1 cDNA was cloned to a cloning and expression vector and two plasmid clones with different ATHL1 CDS insert were obtained. (3) Each plasmid DNA was transformed into Escherichia coli cells for expression and two isoforms of chicken PGGHG were obtained. (4) Both isoforms effectively released glucose from type IV collagen. Next, we searched for carboxyl residues crucial for catalytic activity as follows; human ATHL1 cDNA was cloned into a cloning and expression vector and 18 mutants were obtained by site-directed mutagenesis for 15 carboxyl residues conserved in ATHL1 of jawed vertebrates. The expression analysis indicated that substitutions of Asp301, Glu430 and Glu574 with sterically conservative (D301N, E430Q, E574Q) or functionally conservative (D301E, E430D, E574D) residues led to the complete elimination of enzyme activity. These findings lead us to the conclusion that PGGHG is encoded by ATHL1 and three carboxyl residues (corresponding to Asp301, Glu430 and Glu574 of human PGGHG) might be involved in the catalytic site of PGGHG. Copyright © 2015 Elsevier Inc. All rights reserved.

Toward a Molecular Understanding of Protein Solubility: Increased Negative Surface Charge Correlates with Increased Solubility

PubMed Central

Kramer, Ryan M.; Shende, Varad R.; Motl, Nicole; Pace, C. Nick; Scholtz, J. Martin

2012-01-01

Protein solubility is a problem for many protein chemists, including structural biologists and developers of protein pharmaceuticals. Knowledge about how intrinsic factors influence solubility is limited due to the difficulty of obtaining quantitative solubility measurements. Solubility measurements in buffer alone are difficult to reproduce, because gels or supersaturated solutions often form, making it impossible to determine solubility values for many proteins. Protein precipitants can be used to obtain comparative solubility measurements and, in some cases, estimations of solubility in buffer alone. Protein precipitants fall into three broad classes: salts, long-chain polymers, and organic solvents. Here, we compare the use of representatives from two classes of precipitants, ammonium sulfate and polyethylene glycol 8000, by measuring the solubility of seven proteins. We find that increased negative surface charge correlates strongly with increased protein solubility and may be due to strong binding of water by the acidic amino acids. We also find that the solubility results obtained for the two different precipitants agree closely with each other, suggesting that the two precipitants probe similar properties that are relevant to solubility in buffer alone. PMID:22768947

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

ERIC Educational Resources Information Center

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

2006-01-01

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

Increase in local protein concentration by field-inversion gel electrophoresis.

PubMed

Tsai, Henghang; Low, Teck Yew; Freeby, Steve; Paulus, Aran; Ramnarayanan, Kalpana; Cheng, Chung-Pui Paul; Leung, Hon-Chiu Eastwood

2007-09-26

Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE). Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE. The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.

Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

PubMed

Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T; Day, Kasey J; Keenan, Robert J

2017-07-20

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

Inactivation of a gene that is highly conserved in Gram-positive bacteria stimulates degradation of non-native proteins and concomitantly increases stress tolerance in Lactococcus lactis.

PubMed

Frees, D; Varmanen, P; Ingmer, H

2001-07-01

Exposure of cells to elevated temperatures triggers the synthesis of chaperones and proteases including components of the conserved Clp protease complex. We demonstrated previously that the proteolytic subunit, ClpP, plays a major role in stress tolerance and in the degradation of non-native proteins in the Gram-positive bacterium Lactococcus lactis. Here, we used transposon mutagenesis to generate mutants in which the temperature- and puromycin-sensitive phenotype of a lactococcal clpP null mutant was partly alleviated. In all mutants obtained, the transposon was inserted in the L. lactis trmA gene. When analysing a clpP, trmA double mutant, we found that the expression normally induced from the clpP and dnaK promoters in the clpP mutant was reduced to wild-type level upon introduction of the trmA disruption. Additionally, the degradation of puromycyl-containing polypeptides was increased, suggesting that inactivation of trmA compensates for the absence of ClpP by stimulating an as yet unidentified protease that degrades misfolded proteins. When trmA was disrupted in wild-type cells, both stress tolerance and proteolysis of puromycyl peptides was enhanced above wild-type level. Based on our results, we propose that TrmA, which is well conserved in several Gram-positive bacteria, affects the degradation of non-native proteins and thereby controls stress tolerance.

Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations.

PubMed

Zhao, Jing; Frauenkron-Machedjou, Victorine Josiane; Kardashliev, Tsvetan; Ruff, Anna Joëlle; Zhu, Leilei; Bocola, Marco; Schwaneberg, Ulrich

2017-04-01

The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis. Seventy-six percent of the SeSaM-Tv P/P-generated substitutions yield chemically different amino acid substitutions compared to 64% (epPCR-low) and 69% (epPCR-high). Unique substitutions from one amino acid to others are termed distinct amino acid substitutions. In the SeSaM-Tv P/P library, 35% of all theoretical distinct amino acid substitutions were found in the 1000 mutation library compared to 25% (epPCR-low) and 26% (epPCR-high). Thirty-six percent of distinct amino acid substitutions found in SeSaM-Tv P/P were unobtainable by epPCR-low. Comparison with the SSM library showed that epPCR-low covers 15%, epPCR-high 18%, and SeSaM-Tv P/P 21% of obtainable beneficial amino acid positions. In essence, this study provides first insights on the quality of epPCR and SeSaM-Tv P/P libraries in terms of amino acid substitutions, their chemical differences, and the number of obtainable beneficial amino acid positions.

What Can a Micronucleus Teach? Learning about Environmental Mutagenesis

ERIC Educational Resources Information Center

Linde, Ana R.; Garcia-Vazquez, Eva

2009-01-01

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

Genome-wide comparison of ultraviolet and ethyl methanesulphonate mutagenesis methods for the brown alga Ectocarpus.

PubMed

Godfroy, Olivier; Peters, Akira F; Coelho, Susana M; Cock, J Mark

2015-12-01

Ectocarpus has emerged as a model organism for the brown algae and a broad range of genetic and genomic resources are being generated for this species. The aim of the work presented here was to evaluate two mutagenesis protocols based on ultraviolet irradiation and ethyl methanesulphonate treatment using genome resequencing to measure the number, type and distribution of mutations generated by the two methods. Ultraviolet irradiation generated a greater number of genetic lesions than ethyl methanesulphonate treatment, with more than 400 mutations being detected in the genome of the mutagenised individual. This study therefore confirms that the ultraviolet mutagenesis protocol is suitable for approaches that require a high density of mutations, such as saturation mutagenesis or Targeting Induced Local Lesions in Genomes (TILLING). Copyright © 2015 Elsevier B.V. All rights reserved.

Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses.

PubMed

Rasala, Beth A; Mayfield, Stephen P

2015-03-01

Recombinant proteins are widely used for industrial, nutritional, and medical applications. Green microalgae have attracted considerable attention recently as a biomanufacturing platform for the production of recombinant proteins for a number of reasons. These photosynthetic eukaryotic microorganisms are safe, scalable, easy to genetically modify through transformation, mutagenesis, or breeding, and inexpensive to grow. Many microalgae species are genetically transformable, but the green alga Chlamydomonas reinhardtii is the most widely used host for recombinant protein expression. An extensive suite of molecular genetic tools has been developed for C. reinhardtii over the last 25 years, including a fully sequenced genome, well-established methods for transformation, mutagenesis and breeding, and transformation vectors for high levels of recombinant protein accumulation and secretion. Here, we review recent successes in the development of C. reinhardtii as a biomanufacturing host for recombinant proteins, including antibodies and immunotoxins, hormones, industrial enzymes, an orally-active colostral protein for gastrointestinal health, and subunit vaccines. In addition, we review the biomanufacturing potential of other green algae from the genera Dunaliella and Chlorella.

Recombineering in Streptococcus mutans Using Direct Repeat-Mediated Cloning-Independent Markerless Mutagenesis (DR-CIMM).

PubMed

Zhang, Shan; Zou, Zhengzhong; Kreth, Jens; Merritt, Justin

2017-01-01

Studies of the dental caries pathogen Streptococcus mutans have benefitted tremendously from its sophisticated genetic system. As part of our own efforts to further improve upon the S. mutans genetic toolbox, we previously reported the development of the first cloning-independent markerless mutagenesis (CIMM) system for S. mutans and illustrated how this approach could be adapted for use in many other organisms. The CIMM approach only requires overlap extension PCR (OE-PCR) protocols to assemble counterselectable allelic replacement mutagenesis constructs, and thus greatly increased the speed and efficiency with which markerless mutations could be introduced into S. mutans . Despite its utility, the system is still subject to a couple limitations. Firstly, CIMM requires negative selection with the conditionally toxic phenylalanine analog p -chlorophenylalanine (4-CP), which is efficient, but never perfect. Typically, 4-CP negative selection results in a small percentage of naturally resistant background colonies. Secondly, CIMM requires two transformation steps to create markerless mutants. This can be inherently problematic if the transformability of the strain is negatively impacted after the first transformation step, which is used to insert the counterselection cassette at the mutation site on the chromosome. In the current study, we develop a next-generation counterselection cassette that eliminates 4-CP background resistance and combine this with a new direct repeat-mediated cloning-independent markerless mutagenesis (DR-CIMM) system to specifically address the limitations of the prior approach. DR-CIMM is even faster and more efficient than CIMM for the creation of all types of deletions, insertions, and point mutations and is similarly adaptable for use in a wide range of genetically tractable bacteria.

TALEN-mediated targeted mutagenesis of fatty acid desaturase 2 (FAD2) in peanut (Arachis hypogaea L.) promotes the accumulation of oleic acid.

PubMed

Wen, Shijie; Liu, Hao; Li, Xingyu; Chen, Xiaoping; Hong, Yanbin; Li, Haifen; Lu, Qing; Liang, Xuanqiang

2018-05-01

A first creation of high oleic acid peanut varieties by using transcription activator-like effecter nucleases (TALENs) mediated targeted mutagenesis of Fatty Acid Desaturase 2 (FAD2). Transcription activator like effector nucleases (TALENs), which allow the precise editing of DNA, have already been developed and applied for genome engineering in diverse organisms. However, they are scarcely used in higher plant study and crop improvement, especially in allopolyploid plants. In the present study, we aimed to create targeted mutagenesis by TALENs in peanut. Targeted mutations in the conserved coding sequence of Arachis hypogaea fatty acid desaturase 2 (AhFAD2) were created by TALENs. Genetic stability of AhFAD2 mutations was identified by DNA sequencing in up to 9.52 and 4.11% of the regeneration plants at two different targeted sites, respectively. Mutation frequencies among AhFAD2 mutant lines were significantly correlated to oleic acid accumulation. Genetically, stable individuals of positive mutant lines displayed a 0.5-2 fold increase in the oleic acid content compared with non-transgenic controls. This finding suggested that TALEN-mediated targeted mutagenesis could increase the oleic acid content in edible peanut oil. Furthermore, this was the first report on peanut genome editing event, and the obtained high oleic mutants could serve for peanut breeding project.

Genome-Wide Mutagenesis in Borrelia burgdorferi.

PubMed

Lin, Tao; Gao, Lihui

2018-01-01

Signature-tagged mutagenesis (STM) is a functional genomics approach to identify bacterial virulence determinants and virulence factors by simultaneously screening multiple mutants in a single host animal, and has been utilized extensively for the study of bacterial pathogenesis, host-pathogen interactions, and spirochete and tick biology. The signature-tagged transposon mutagenesis has been developed to investigate virulence determinants and pathogenesis of Borrelia burgdorferi. Mutants in genes important in virulence are identified by negative selection in which the mutants fail to colonize or disseminate in the animal host and tick vector. STM procedure combined with Luminex Flex ® Map™ technology and next-generation sequencing (e.g., Tn-seq) are the powerful high-throughput tools for the determination of Borrelia burgdorferi virulence determinants. The assessment of multiple tissue sites and two DNA resources at two different time points using Luminex Flex ® Map™ technology provides a robust data set. B. burgdorferi transposon mutant screening indicates that a high proportion of genes are the novel virulence determinants that are required for mouse and tick infection. In this protocol, an effective signature-tagged Himar1-based transposon suicide vector was developed and used to generate a sequence-defined library of nearly 4800 mutants in the infectious B. burgdorferi B31 clone. In STM, signature-tagged suicide vectors are constructed by inserting unique DNA sequences (tags) into the transposable elements. The signature-tagged transposon mutants are generated when transposon suicide vectors are transformed into an infectious B. burgdorferi clone, and the transposable element is transposed into the 5'-TA-3' sequence in the B. burgdorferi genome with the signature tag. The transposon library is created and consists of many sub-libraries, each sub-library has several hundreds of mutants with same tags. A group of mice or ticks are infected with a mixed

A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA.

PubMed

Minkoff, Benjamin B; Makino, Shin-Ichi; Haruta, Miyoshi; Beebe, Emily T; Wrobel, Russell L; Fox, Brian G; Sussman, Michael R

2017-04-07

There are more than 600 receptor-like kinases (RLKs) in Arabidopsis , but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

CYTOTOXICITY AND MUTAGENESIS METHODS FOR EVALUATING TOXICITY REMOVAL FROM WASTEWATERS

EPA Science Inventory

This project was a feasibility study of the effectiveness of a mammalian cell cytotoxicity assay and a mammalian cell mutagenesis assay for monitoring the toxicity and mutagenicity of influent and effluent wastewater at treatment plants. In the cytotoxicity assay, ambient samples...

Application of In Vitro Transposon Mutagenesis to Erythromycin Strain Improvement in Saccharopolyspora erythraea.

PubMed

Weber, J Mark; Reeves, Andrew; Cernota, William H; Wesley, Roy K

2017-01-01

Transposon mutagenesis is an invaluable technique in molecular biology for the creation of random mutations that can be easily identified and mapped. However, in the field of microbial strain improvement, transposon mutagenesis has scarcely been used; instead, chemical and physical mutagenic methods have been traditionally favored. Transposons have the advantage of creating single mutations in the genome, making phenotype to genotype assignments less challenging than with traditional mutagens which commonly create multiple mutations in the genome. The site of a transposon mutation can also be readily mapped using DNA sequencing primer sites engineered into the transposon termini. In this chapter an in vitro method for transposon mutagenesis of Saccharopolyspora erythraea is presented. Since in vivo transposon tools are not available for most actinomycetes including S. erythraea, an in vitro method was developed. The in vitro method involves a significant investment in time and effort to create the mutants, but once the mutants are made and screened, a large number of highly relevant mutations of direct interest to erythromycin production can be found.

Demonstration of Lignin-to-Peroxidase Direct Electron Transfer: A TRANSIENT-STATE KINETICS, DIRECTED MUTAGENESIS, EPR, AND NMR STUDY.

PubMed

Sáez-Jiménez, Verónica; Baratto, Maria Camilla; Pogni, Rebecca; Rencoret, Jorge; Gutiérrez, Ana; Santos, José Ignacio; Martínez, Angel T; Ruiz-Dueñas, Francisco Javier

2015-09-18

Versatile peroxidase (VP) is a high redox-potential peroxidase of biotechnological interest that is able to oxidize phenolic and non-phenolic aromatics, Mn(2+), and different dyes. The ability of VP from Pleurotus eryngii to oxidize water-soluble lignins (softwood and hardwood lignosulfonates) is demonstrated here by a combination of directed mutagenesis and spectroscopic techniques, among others. In addition, direct electron transfer between the peroxidase and the lignin macromolecule was kinetically characterized using stopped-flow spectrophotometry. VP variants were used to show that this reaction strongly depends on the presence of a solvent-exposed tryptophan residue (Trp-164). Moreover, the tryptophanyl radical detected by EPR spectroscopy of H2O2-activated VP (being absent from the W164S variant) was identified as catalytically active because it was reduced during lignosulfonate oxidation, resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation at 355 nm/emission at 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in one-dimensional and two-dimensional NMR spectra, confirming the ligninolytic capabilities of the enzyme. Simultaneously, size-exclusion chromatography showed an increase of the molecular mass of the modified residual lignin, especially for the (low molecular mass) hardwood lignosulfonate, revealing that the oxidation products tend to recondense during the VP treatment. Finally, mutagenesis of selected residues neighboring Trp-164 resulted in improved apparent second-order rate constants for lignosulfonate reactions, revealing that changes in its protein environment (modifying the net negative charge and/or substrate accessibility/binding) can modulate the reactivity of the catalytic tryptophan. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Transponson Tn916 Mutagenesis in Bacillus anthracis,

DTIC Science & Technology

1987-11-10

Tngla, is described. Tng1a was transferred from Streptococcus 1aJaji strain DS16C1 to f. a VNR-1 by conjugation in a standard filter mating procedure...transposon, Tn916, mutagenesis, Bacillus, anthracis, subtilis. , Streptococcus , faecalis, aro 2.AUSrN ACT (Cautious no reverse efho if nece.at7r sd ideratfy...transferred from Streptococcus : faecalis strain DS16CI to B. anthracis VNR-1 by conjugation in a standard filter mating procedure. Tetracycline

Environmental mutagenesis during the end-Permian ecological crisis.

PubMed

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

2004-08-31

During the end-Permian ecological crisis, terrestrial ecosystems experienced preferential dieback of woody vegetation. Across the world, surviving herbaceous lycopsids played a pioneering role in repopulating deforested terrain. We document that the microspores of these lycopsids were regularly released in unseparated tetrads indicative of failure to complete the normal process of spore development. Although involvement of mutation has long been hinted at or proposed in theory, this finding provides concrete evidence for chronic environmental mutagenesis at the time of global ecological crisis. Prolonged exposure to enhanced UV radiation could account satisfactorily for a worldwide increase in land plant mutation. At the end of the Permian, a period of raised UV stress may have been the consequence of severe disruption of the stratospheric ozone balance by excessive emission of hydrothermal organohalogens in the vast area of Siberian Traps volcanism. Copyright 2004 The National Academy of Sciencs of the USA

NET1 and HFI1 genes of yeast mediate both chromosome maintenance and mitochondrial rho(-) mutagenesis.

PubMed

Koltovaya, N A; Guerasimova, A S; Tchekhouta, I A; Devin, A B

2003-08-01

An increase in the mitochondrial rho(-) mutagenesis is a well-known response of yeast cells to mutations in numerous nuclear genes as well as to various kinds of stress. Despite extensive studies for several decades, the biological significance of this response is still not fully understood. The genetic approach to solving this enigma includes a study of genes that are required for the high incidence of spontaneous rho(-) mutants. We have obtained mutations of a few nuclear genes of that sort and found that mutations in certain genes, including CDC28, the central cell-cycle regulation gene, result in a decrease in spontaneous rho(-) mutability and simultaneously affect the maintenance of the yeast chromosomes and plasmids. Two more genes resembling CDC28 in this respect are identified in the present work as a result of the characterization of four new mutants. These two genes are NET1 and HFI1 which mediate important regulatory protein-protein interactions in the yeast cell. The effects of four mutations, including net1-srm and hfi1-srm, on the maintenance of the yeast mitochondrial genome, chromosomes and plasmids, as well as on the cell's sensitivity to ionizing radiation, are also described. The data presented suggest that the pleiotropic srm mutations determining coordinate changes in the fidelity of mitotic transmission of chromosomes, plasmids and mtDNA molecules identify genes that most probably operate high up in the hierarchy of the general genetic regulation of yeast. Copyright 2003 John Wiley & Sons, Ltd.

Shortening a loop can increase protein native state entropy.

PubMed

Gavrilov, Yulian; Dagan, Shlomi; Levy, Yaakov

2015-12-01

Protein loops are essential structural elements that influence not only function but also protein stability and folding rates. It was recently reported that shortening a loop in the AcP protein may increase its native state conformational entropy. This effect on the entropy of the folded state can be much larger than the lower entropic penalty of ordering a shorter loop upon folding, and can therefore result in a more pronounced stabilization than predicted by polymer model for loop closure entropy. In this study, which aims at generalizing the effect of loop length shortening on native state dynamics, we use all-atom molecular dynamics simulations to study how gradual shortening a very long or solvent-exposed loop region in four different proteins can affect their stability. For two proteins, AcP and Ubc7, we show an increase in native state entropy in addition to the known effect of the loop length on the unfolded state entropy. However, for two permutants of SH3 domain, shortening a loop results only with the expected change in the entropy of the unfolded state, which nicely reproduces the observed experimental stabilization. Here, we show that an increase in the native state entropy following loop shortening is not unique to the AcP protein, yet nor is it a general rule that applies to all proteins following the truncation of any loop. This modification of the loop length on the folded state and on the unfolded state may result with a greater effect on protein stability. © 2015 Wiley Periodicals, Inc.

Combinatorial effect of mutagenesis and medium component optimization on Bacillus amyloliquefaciens antifungal activity and efficacy in eradicating Botrytis cinerea.

PubMed

Masmoudi, Fatma; Ben Khedher, Saoussen; Kamoun, Amel; Zouari, Nabil; Tounsi, Slim; Trigui, Mohamed

2017-04-01

This work is directed towards Bacillus amyloliquefaciens strain BLB371 metabolite production for biocontrol of fungal phytopathogens. In order to maximise antifungal metabolite production by this strain, two approaches were combined: random mutagenesis and medium component optimization. After three rounds of mutagenesis, a hyper active mutant, named M3-7, was obtained. It produces 7 fold more antifungal metabolites (1800AU/mL) than the wild strain in MC medium. A hybrid design was applied to optimise a new medium to enhance antifungal metabolite production by M3-7. The new optimized medium (35g/L of peptone, 32.5g/L of sucrose, 10.5g/L of yeast extract, 2.4g/L of KH 2 PO 4 , 1.3g/L of MgSO 4 and 23mg/L of MnSO 4 ) achieved 1.62 fold enhancement in antifungal compound production (3000AU/mL) by this mutant, compared to that achieved in MC medium. Therefore, combinatory effect of these two approaches (mutagenesis and medium component optimization) allowed 12 fold improvement in antifungal activity (from 250UA/mL to 3000UA/mL). This improvement was confirmed against several phytopathogenic fungi with an increase of MIC and MFC over than 50%. More interestingly, a total eradication of gray mold was obtained on tomato fruits infected by Botrytis cinerea and treated by M3-7, compared to those treated by BLB371. From the practical point of view, combining random mutagenesis and medium optimization could be considered as an excellent tool for obtaining promising biological products useful against phytopathogenic fungi. Copyright © 2017 Elsevier GmbH. All rights reserved.

Chromophore photophysics and dynamics in fluorescent proteins of the GFP family

NASA Astrophysics Data System (ADS)

Nienhaus, Karin; Nienhaus, G. Ulrich

2016-11-01

Proteins of the green fluorescent protein (GFP) family are indispensable for fluorescence imaging experiments in the life sciences, particularly of living specimens. Their essential role as genetically encoded fluorescence markers has motivated many researchers over the last 20 years to further advance and optimize these proteins by using protein engineering. Amino acids can be exchanged by site-specific mutagenesis, starting with naturally occurring proteins as templates. Optical properties of the fluorescent chromophore are strongly tuned by the surrounding protein environment, and a targeted modification of chromophore-protein interactions requires a profound knowledge of the underlying photophysics and photochemistry, which has by now been well established from a large number of structural and spectroscopic experiments and molecular-mechanical and quantum-mechanical computations on many variants of fluorescent proteins. Nevertheless, such rational engineering often does not meet with success and thus is complemented by random mutagenesis and selection based on the optical properties. In this topical review, we present an overview of the key structural and spectroscopic properties of fluorescent proteins. We address protein-chromophore interactions that govern ground state optical properties as well as processes occurring in the electronically excited state. Special emphasis is placed on photoactivation of fluorescent proteins. These light-induced reactions result in large structural changes that drastically alter the fluorescence properties of the protein, which enables some of the most exciting applications, including single particle tracking, pulse chase imaging and super-resolution imaging. We also present a few examples of fluorescent protein application in live-cell imaging experiments.

Identification of NH4+-regulated genes of Herbaspirillum seropedicae by random insertional mutagenesis.

PubMed

Schwab, Stefan; Ramos, Humberto J; Souza, Emanuel M; Pedrosa, Fábio O; Yates, Marshall G; Chubatsu, Leda S; Rigo, Liu U

2007-05-01

Random mutagenesis using transposons with promoterless reporter genes has been widely used to examine differential gene expression patterns in bacteria. Using this approach, we have identified 26 genes of the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae regulated in response to ammonium content in the growth medium. These include nine genes involved in the transport of nitrogen compounds, such as the high-affinity ammonium transporter AmtB, and uptake systems for alternative nitrogen sources; nine genes coding for proteins responsible for restoring intracellular ammonium levels through enzymatic reactions, such as nitrogenase, amidase, and arginase; and a third group includes metabolic switch genes, coding for sensor kinases or transcription regulation factors, whose role in metabolism was previously unknown. Also, four genes identified were of unknown function. This paper describes their involvement in response to ammonium limitation. The results provide a preliminary profile of the metabolic response of Herbaspirillum seropedicae to ammonium stress.

[Control levels of Sin3 histone deacetylase for spontaneous and UV-induced mutagenesis in yeasts Saccharomyces cerevisiae].

PubMed

Lebovka, I Iu; Kozhina, T N; Fedorova, I V; Peshekhonov, V T; Evstiukhina, T A; Chernenkov, A Iu; Korolev, V G

2014-01-01

SIN3 gene product operates as a repressor for a huge amount of genes in Saccharomyces cerevisiae. Sin3 protein with a mass of about 175 kDa is a member of the RPD3 protein complex with an assessed mass of greater than 2 million Da. It was previously shownthat RPD3 gene mutations influence recombination and repair processes in S. cerevisiae yeasts. We studied the impacts of the sin3 mutation on UV-light sensitivity and UV-induced mutagenesis in budding yeast cells. The deletion ofthe SIN3 gene causes weak UV-sensitivity of mutant budding cells as compared to the wild-type strain. These results show that the sin3 mutation decreases both spontaneous and UV-induced levels of levels. This fact is hypothetically related to themalfunction of ribonucleotide reductase activity regulation, which leads to a decrease in the dNTP pool and the inaccurate error-prone damage bypass postreplication repair pathway, which in turn provokes a reduction in the incidence of mutations.

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

PubMed Central

Hall, Robyn N.; Meers, Joanne; Fowler, Elizabeth; Mahony, Timothy

2012-01-01

Bacterial artificial chromosome (BAC) vectors were first developed to facilitate the propagation and manipulation of large DNA fragments in molecular biology studies for uses such as genome sequencing projects and genetic disease models. To facilitate these studies, methodologies have been developed to introduce specific mutations that can be directly applied to the mutagenesis of infectious clones (icBAC) using BAC technologies. This has resulted in rapid identification of gene function and expression at unprecedented rates. Here we review the major developments in BAC mutagenesis in vitro. This review summarises the technologies used to construct and introduce mutations into herpesvirus icBAC. It also explores developing technologies likely to provide the next leap in understanding these important viruses. PMID:22470833

Genetic Regulation of Charged Particle Mutagenesis in Human Cells

NASA Technical Reports Server (NTRS)

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

1999-01-01

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

The rice blast resistance gene Ptr encodes an atypical protein required for broad spectrum disease resistance

USDA-ARS?s Scientific Manuscript database

Plant resistance (R) genes typically encode proteins with nucleotide binding site-leucine rich repeat (NLR) domains. We identified a novel, broad-spectrum rice blast R gene, Ptr, encoding a non-NLR protein with four Armadillo repeats. Ptr was originally identified by fast neutron mutagenesis as a ...

Radiation induced COX-2 expression and mutagenesis at non-targeted lung tissues of gpt delta transgenic mice

PubMed Central

Chai, Y; Calaf, G M; Zhou, H; Ghandhi, S A; Elliston, C D; Wen, G; Nohmi, T; Amundson, S A; Hei, T K

2013-01-01

Background: Although radiation-induced bystander effects have been confirmed using a variety of endpoints, the mechanism(s) underlying these effects are not well understood, especially for in vivo study. Methods: A 1-cm2 area (1 cm × 1 cm) in the lower abdominal region of gpt delta transgenic mice was irradiated with 5 Gy of 300 keV X-rays, and changes in out-of-field lung and liver were observed. Results: Compared with sham-treated controls, the Spi− mutation frequency increased 2.4-fold in non-targeted lung tissues at 24 h after partial body irradiation (PBIR). Consistent with dramatic Cyclooxygenase 2 (COX-2) induction in the non-targeted bronchial epithelial cells, increasing levels of prostaglandin, together with 8-hydroxydeoxyguanosine, in the out-of-field lung tissues were observed after PBIR. In addition, DNA double-strand breaks and apoptosis were induced in bystander lung tissues after PBIR. Conclusion: The PBIR induces DNA damage and mutagenesis in non-targeted lung tissues, especially in bronchial epithelial cells, and COX-2 has an essential role in bystander mutagenesis. PMID:23321513

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

PubMed

Kristensen, Tatjana P; Maria Cherian, Reeja; Gray, Fiona C; MacNeill, Stuart A

2014-01-01

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

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

PubMed Central

Kristensen, Tatjana P.; Maria Cherian, Reeja; Gray, Fiona C.; MacNeill, Stuart A.

2014-01-01

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies. PMID:24723920

Transcriptome analysis of Neisseria meningitidis in human whole blood and mutagenesis studies identify virulence factors involved in blood survival.

PubMed

Echenique-Rivera, Hebert; Muzzi, Alessandro; Del Tordello, Elena; Seib, Kate L; Francois, Patrice; Rappuoli, Rino; Pizza, Mariagrazia; Serruto, Davide

2011-05-01

During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by

Transcriptome Analysis of Neisseria meningitidis in Human Whole Blood and Mutagenesis Studies Identify Virulence Factors Involved in Blood Survival

PubMed Central

Del Tordello, Elena; Seib, Kate L.; Francois, Patrice; Rappuoli, Rino; Pizza, Mariagrazia; Serruto, Davide

2011-01-01

During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by

Protein Enrichment of Familiar Foods as an Innovative Strategy to Increase Protein Intake in Institutionalized Elderly.

PubMed

Beelen, J; de Roos, N M; de Groot, L C P G M

2017-01-01

To increase the protein intake of older adults, protein enrichment of familiar foods and drinks might be an effective and attractive alternative for oral nutritional supplements (ONS). We performed a pilot study to test whether these products could help institutionalized elderly to reach a protein intake of 1.2 gram per kg body weight per day (g/kg/d). Intervention study with one treatment group (no control group). Dietary assessment was done before and at the end of a 10-day intervention. Two care facilities in Gelderland, the Netherlands: a residential care home and a rehabilitation center. 22 elderly subjects (13 women, 9 men; mean age 83.0±9.4 years). We used a variety of newly developed protein enriched regular foods and drinks, including bread, soups, fruit juices, and instant mashed potatoes. Dietary intake was assessed on two consecutive days before and at the end of the intervention, using food records filled out by research assistants. Energy and macronutrient intake was calculated using the 2013 Dutch food composition database. Changes in protein intake were evaluated using paired t-tests. Protein intake increased by 11.8 g/d (P=0.003); from 0.96 to 1.14 g/kg/d (P=0.002). This increase is comparable to protein provided by one standard portion of ONS. The intake of energy and other macronutrients did not change significantly. At the end of the intervention more elderly reached a protein intake level of 1.2 g/kg/d than before (9 vs 4). Protein intake significantly increased during breakfast (+3.7 g) and during the evening (+2.2 g). Including familiar protein enriched foods and drinks in the menu helped to meet protein recommendations in institutionalized elderly.

Molecular contacts in the transmembrane c-subunit oligomer of F-ATPases identified by tryptophan substitution mutagenesis.

PubMed

Schnick, C; Forrest, L R; Sansom, M S; Groth, G

2000-07-20

When isolated in its monomeric form, subunit c of the proton transporting ATP synthase of Escherichia coli was shown to fold in a hairpin-like structure consisting of two hydrophobic membrane spanning helices and a short connecting hydrophilic loop. In the plasma membrane of Escherichia coli, however, about 9-12 c-subunit monomers form an oligomeric complex that functions in transmembrane proton conduction and in energy transduction to the catalytic F1 domain. The arrangement of the monomers and the molecular architecture of the complex were studied by tryptophan scanning mutagenesis and restrained MD simulations. Residues 12-24 of the N-terminal transmembrane segment of subunit c were individually substituted by the large and moderately hydrophobic tryptophan side chain. Effects on the activity of the mutant proteins were studied in selective growth experiments and various ATP synthase specific activity assays. The results identify potential intersubunit contacts and structurally non-distorted, accessible residues in the c-oligomer and add constraints to the arrangement of monomers in the oligomeric complex. Results from our mutagenesis experiments were interpreted in structural models of the c-oligomer that have been obtained by restrained MD simulations. Different stoichiometries and monomer orientations were applied in these calculations. A cylindrical complex consisting of 10 monomers that are arranged in two concentric rings with the N-terminal helices of the monomers located at the periphery shows the best match with the experimental data.

Protein carboxyl methylation increases in parallel with differentiation of neuroblastoma cells.

PubMed

Kloog, Y; Axelrod, J; Spector, I

1983-02-01

Cells of mouse neuroblastoma clone N1E-115 in the confluent phase of growth can catalyze the formation of endogenous protein carboxyl methyl esters, using a protein carboxyl methylase and membrane-bound methyl acceptor proteins. The enzyme is localized predominantly in the cytosol of the cells and has a molecular weight of about 20,000 daltons. Treatment of the cells with dimethylsulfoxide (DMSO) or hexamethylene-bisacetamide (HMBA), agents that induce morphological and electrophysiological differentiation, results in a marked increase in protein carboxyl methylase activity. Maximal levels are reached 6-7 days after exposure to the agents, a time course that closely parallels the development of electrical excitability mechanisms in these cells. Serum deprivation also causes neurite outgrowth but does not enhance electrical excitability or enzyme activity. The capacity of membrane-bound neuroblastoma protein(s) to be carboxyl methylated is increased by the differentiation procedures that have been examined. However, the increase in methyl acceptor proteins induced by DMSO or HMBA is the largest, and its time course parallels electrophysiological differentiation. In contrast, serum deprivation induced a small increase that reached maximal levels within 24 h. The data suggest that increased protein carboxyl methylation is a developmentally regulated property of neuroblastoma cells and that at least two groups of methyl acceptor proteins are induced during differentiation: a minor group related to morphological differentiation, and a major group that may be related to ionic permeability mechanisms of the excitable membrane.

Acute toxicity and mutagenesis of three metabolites mixture of nitrobenzene in mice.

PubMed

Wang, Guixia; Zhang, Xiuying; Yao, Chunzhu; Tian, Meizhan

2011-03-01

Nitrobenzene is a synthetic compound, more than 95% of which is used in the production of aniline. Nitrobenzene has been demonstrated to be substantially metabolized to p-Nitrophenol, p-Aminophenol and p-Nitroaniline in food animals (e.g., bovines, fowls). There have been no studies on the acute toxicity and the mutagenesis of the mixture of the three metabolites mentioned above. The aim of the present study is to testify the acute toxicity and the mutagenesis of the three metabolites mixture. Seventy Kunming mice (half male, half female) received an intragastric administration exposure to metabolites-containing suspension of 750, 638, 542, 461, 392, 333 mg kg(-1) body weight and 0.5% sodium carboxymethyl cellulose (control), followed by a 14-day observation. The medial lethal dose (LD(50)) concentration for nitrobenzene metabolites mixture in this study was 499.92 mg/kg. Their mutagenic toxicology was studied through micronucleus and sperm abnormality test. Kunming mice were twice intragastrically exposed to 1/5 LD(50), 1/10 LD(50), 1/20 LD(50) mg kg(-1) nitrobenzene metabolites-containing suspension spaced 24-h apart. Cyclophosphamide, pure water and sodium carboxymethyl cellulose served as doses of the positive group, the negative group and the solvent control group, respectively. The incidence of micronucleus and sperm abnormality increased significantly in the 1/5 LD(50) and 1/10 LD(50) group compared with the negative and solvent control group. A dose-related increase in the incidence of micronucleus and sperm abnormality was noted. In conclusion, the three metabolites mixture of nitrobenzene was secondary toxicity and mutagenic substances in mice.

Antenatal dietary advice and supplementation to increase energy and protein intake.

PubMed

Ota, Erika; Tobe-Gai, Ruoyan; Mori, Rintaro; Farrar, Diane

2012-09-12

Gestational weight gain is positively associated with fetal growth, and observational studies of food supplementation in pregnancy have reported increases in gestational weight gain and fetal growth. To assess the effects of advice during pregnancy to increase energy and protein intake, or of actual energy and protein supplementation, on energy and protein intakes, and the effect on maternal and infant health outcomes. We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (22 July 2011) and contacted researchers in the field. We updated the search on 12 July 2012 and added the results to the awaiting classification section of the review. Randomised controlled trials of dietary advice to increase energy and protein intake, or of actual energy and protein supplementation, during pregnancy. Two review authors independently assessed trials for inclusion and assessed risk of bias. Two review authors independently extracted data and checked for accuracy. Extracted data were supplemented by additional information from the trialists we contacted. We examined 110 reports corresponding to 46 trials. Of these trials, 15 were included, 30 were excluded, and one is ongoing. Overall, 15 trials involving 7410 women were included.Nutritional advice (four trials, 790 women)Women given nutritional advice had a lower relative risk of having a preterm birth (two trials, 449 women) (risk ratio (RR) 0.46, 95% CI 0.21 to 0.98 ), head circumference at birth was increased in one trial (389 women) (mean difference (MD) 0.99 cm, 95% CI 0.43 to 1.55) and protein intake increased (three trials, 632 women) (protein intake: MD +6.99 g/day, 95% CI 3.02 to 10.97). No significant differences were observed on any other outcomes.Balanced energy and protein supplementation (11 trials, 5385 women)Risk of stillbirth was significantly reduced for women given balanced energy and protein supplementation (RR 0.62, 95% CI 0.40 to 0.98, five trials, 3408 women), mean birthweight was

Triplex technology in studies of DNA damage, DNA repair, and mutagenesis.

PubMed

Mukherjee, Anirban; Vasquez, Karen M

2011-08-01

Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

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

PubMed

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

2014-04-01

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

Random mutagenesis of the hyperthermophilic archaeon Pyrococcus furiosus using in vitro mariner transposition and natural transformation.

PubMed

Guschinskaya, Natalia; Brunel, Romain; Tourte, Maxime; Lipscomb, Gina L; Adams, Michael W W; Oger, Philippe; Charpentier, Xavier

2016-11-08

Transposition mutagenesis is a powerful tool to identify the function of genes, reveal essential genes and generally to unravel the genetic basis of living organisms. However, transposon-mediated mutagenesis has only been successfully applied to a limited number of archaeal species and has never been reported in Thermococcales. Here, we report random insertion mutagenesis in the hyperthermophilic archaeon Pyrococcus furiosus. The strategy takes advantage of the natural transformability of derivatives of the P. furiosus COM1 strain and of in vitro Mariner-based transposition. A transposon bearing a genetic marker is randomly transposed in vitro in genomic DNA that is then used for natural transformation of P. furiosus. A small-scale transposition reaction routinely generates several hundred and up to two thousands transformants. Southern analysis and sequencing showed that the obtained mutants contain a single and random genomic insertion. Polyploidy has been reported in Thermococcales and P. furiosus is suspected of being polyploid. Yet, about half of the mutants obtained on the first selection are homozygous for the transposon insertion. Two rounds of isolation on selective medium were sufficient to obtain gene conversion in initially heterozygous mutants. This transposition mutagenesis strategy will greatly facilitate functional exploration of the Thermococcales genomes.

Microgravity protein crystallization

PubMed Central

McPherson, Alexander; DeLucas, Lawrence James

2015-01-01

Over the past 20 years a variety of technological advances in X-ray crystallography have shortened the time required to determine the structures of large macromolecules (i.e., proteins and nucleic acids) from several years to several weeks or days. However, one of the remaining challenges is the ability to produce diffraction-quality crystals suitable for a detailed structural analysis. Although the development of automated crystallization systems combined with protein engineering (site-directed mutagenesis to enhance protein solubility and crystallization) have improved crystallization success rates, there remain hundreds of proteins that either cannot be crystallized or yield crystals of insufficient quality to support X-ray structure determination. In an attempt to address this bottleneck, an international group of scientists has explored use of a microgravity environment to crystallize macromolecules. This paper summarizes the history of this international initiative along with a description of some of the flight hardware systems and crystallization results. PMID:28725714

A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function.

PubMed

Trubiroha, A; Gillotay, P; Giusti, N; Gacquer, D; Libert, F; Lefort, A; Haerlingen, B; De Deken, X; Opitz, R; Costagliola, S

2018-04-04

The foregut endoderm gives rise to several organs including liver, pancreas, lung and thyroid with important roles in human physiology. Understanding which genes and signalling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to develop a rapid and scalable CRISPR/Cas-based mutagenesis strategy aiming at the identification of genes involved in morphogenesis and function of the thyroid. Core elements of the mutagenesis assay comprise bi-allelic gene invalidation in somatic mutants, a non-invasive monitoring of thyroid development in live transgenic fish, complementary analyses of thyroid function in fixed specimens and quantitative analyses of mutagenesis efficiency by Illumina sequencing of individual fish. We successfully validated our mutagenesis-phenotyping strategy in experiments targeting genes with known functions in early thyroid morphogenesis (pax2a, nkx2.4b) and thyroid functional differentiation (duox, duoxa, tshr). We also demonstrate that duox and duoxa crispants phenocopy thyroid phenotypes previously observed in human patients with bi-allelic DUOX2 and DUOXA2 mutations. The proposed combination of efficient mutagenesis protocols, rapid non-invasive phenotyping and sensitive genotyping holds great potential to systematically characterize the function of larger candidate gene panels during thyroid development and is applicable to other organs and tissues.

Estrogen Drives Cellular Transformation and Mutagenesis in Cells Expressing the Breast Cancer-Associated R438W DNA Polymerase Lambda Protein.

PubMed

Nemec, Antonia A; Bush, Korie B; Towle-Weicksel, Jamie B; Taylor, B Frazier; Schulz, Vincent; Weidhaas, Joanne B; Tuck, David P; Sweasy, Joann B

2016-11-01

Repair of DNA damage is critical for maintaining the genomic integrity of cells. DNA polymerase lambda (POLL/Pol λ) is suggested to function in base excision repair (BER) and nonhomologous end-joining (NHEJ), and is likely to play a role in damage tolerance at the replication fork. Here, using next-generation sequencing, it was discovered that the POLL rs3730477 single-nucleotide polymorphism (SNP) encoding R438W Pol λ was significantly enriched in the germlines of breast cancer patients. Expression of R438W Pol λ in human breast epithelial cells induces cellular transformation and chromosomal aberrations. The role of estrogen was assessed as it is commonly used in hormone replacement therapies and is a known breast cancer risk factor. Interestingly, the combination of estrogen treatment and the expression of the R438W Pol λ SNP drastically accelerated the rate of transformation. Estrogen exposure produces 8-oxoguanine lesions that persist in cells expressing R438W Pol λ compared with wild-type (WT) Pol λ-expressing cells. Unlike WT Pol λ, which performs error-free bypass of 8-oxoguanine lesions, expression of R438W Pol λ leads to an increase in mutagenesis and replicative stress in cells treated with estrogen. Together, these data suggest that individuals who carry the rs3730477 POLL germline variant have an increased risk of estrogen-associated breast cancer. The Pol λ R438W mutation can serve as a biomarker to predict cancer risk and implicates that treatment with estrogen in individuals with this mutation may further increase their risk of breast cancer. Mol Cancer Res; 14(11); 1068-77. ©2016 AACR. ©2016 American Association for Cancer Research.

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

PubMed

Buszczak, Michael; Paterno, Shelley; Lighthouse, Daniel; Bachman, Julia; Planck, Jamie; Owen, Stephenie; Skora, Andrew D; Nystul, Todd G; Ohlstein, Benjamin; Allen, Anna; Wilhelm, James E; Murphy, Terence D; Levis, Robert W; Matunis, Erika; Srivali, Nahathai; Hoskins, Roger A; Spradling, Allan C

2007-03-01

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.

The Carnegie Protein Trap Library: A Versatile Tool for Drosophila Developmental Studies

PubMed Central

Buszczak, Michael; Paterno, Shelley; Lighthouse, Daniel; Bachman, Julia; Planck, Jamie; Owen, Stephenie; Skora, Andrew D.; Nystul, Todd G.; Ohlstein, Benjamin; Allen, Anna; Wilhelm, James E.; Murphy, Terence D.; Levis, Robert W.; Matunis, Erika; Srivali, Nahathai; Hoskins, Roger A.; Spradling, Allan C.

2007-01-01

Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600–900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions. PMID:17194782

Application of signature-tagged mutagenesis to the study of virulence of Erwinia amylovora.

PubMed

Wang, Limei; Beer, Steven V

2006-12-01

To identify genes that contribute to the virulence of Erwinia amylovora in plants, 1892 mutants were created and screened in pools of < or =96 mutants using signature-tagged mutagenesis. Nineteen mutants were not recovered from apple shoots following inoculation, which suggested that the insertions in these mutants affected genes important for bacterial survival in planta. DNA flanking the Tn5 insertions in the 19 mutants was sequenced and analysed by blast. One mutant had a Tn5 insertion in amsE, a gene involved in the biosynthesis of exopolysaccaride (EPS). Fourteen mutants had insertions in loci that were implicated in biosynthesis or transport of particular amino acids or nucleotides, a site-specific recombinase active during cell division and several putative proteins of unknown function; the flanking DNA of the remaining four mutants lacked significant homology with any DNA in the database. When inoculated individually to hosts, 10 of the 19 mutants caused significantly less disease and multiplied less, as compared with the wild-type strain.

Exploring protein structure and dynamics through a project-oriented biochemistry laboratory module.

PubMed

Lipchock, James M; Ginther, Patrick S; Douglas, Bonnie B; Bird, Kelly E; Patrick Loria, J

2017-09-01

Here, we present a 10-week project-oriented laboratory module designed to provide a course-based undergraduate research experience in biochemistry that emphasizes the importance of biomolecular structure and dynamics in enzyme function. This module explores the impact of mutagenesis on an important active site loop for a biomedically-relevant human enzyme, protein tyrosine phosphatase 1B (PTP1B). Over the course of the semester students guide their own mutant of PTP1B from conception to characterization in a cost-effective manner and gain exposure to fundamental techniques in biochemistry, including site-directed DNA mutagenesis, bacterial recombinant protein expression, affinity column purification, protein quantitation, SDS-PAGE, and enzyme kinetics. This project-based approach allows an instructor to simulate a research setting and prepare students for productive research beyond the classroom. Potential modifications to expand or contract this module are also provided. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):403-410, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

Consumption of Milk Protein or Whey Protein Results in a Similar Increase in Muscle Protein Synthesis in Middle Aged Men

PubMed Central

Mitchell, Cameron J.; McGregor, Robin A.; D’Souza, Randall F.; Thorstensen, Eric B.; Markworth, James F.; Fanning, Aaron C.; Poppitt, Sally D.; Cameron-Smith, David

2015-01-01

The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring 13C6 phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h−1 in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p < 0.001) to 0.057% ± 0.018% and 0.052% ± 0.024% h−1 in the milk and whey groups respectively with no difference between groups (p = 0.810). FSR returned to baseline in both groups between 90 and 210 min after protein ingestion. Despite evidence of increased rate of digestion and leucine availability following the ingestion of whey protein, there was similar activation of MPS in middle-aged men with either 20 g of milk protein or whey protein. PMID:26506377

STD-NMR-Based Protein Engineering of the Unique Arylpropionate-Racemase AMDase G74C.

PubMed

Gaßmeyer, Sarah Katharina; Yoshikawa, Hiroyuki; Enoki, Junichi; Hülsemann, Nadine; Stoll, Raphael; Miyamoto, Kenji; Kourist, Robert

2015-06-23

Structure-guided protein engineering achieved a variant of the unique racemase AMDase G74C, with 40-fold increased activity in the racemisation of several arylaliphatic carboxylic acids. Substrate binding during catalysis was investigated by saturation-transfer-difference NMR (STD-NMR) spectroscopy. All atoms of the substrate showed interactions with the enzyme. STD-NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose substitutions increased the activity of G74C. Single amino acid exchanges increased the activity moderately; structure-guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD-NMR as versatile tool for the analysis of enzyme-substrate interactions in catalytically competent systems and for the guidance of protein engineering. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bacteroides fragilis RecA protein overexpression causes resistance to metronidazole

PubMed Central

Steffens, Laura S.; Nicholson, Samantha; Paul, Lynthia V.; Nord, Carl Erik; Patrick, Sheila; Abratt, Valerie R.

2010-01-01

Bacteroides fragilis is a human gut commensal and an opportunistic pathogen causing anaerobic abscesses and bacteraemias which are treated with metronidazole (Mtz), a DNA damaging agent. This study examined the role of the DNA repair protein, RecA, in maintaining endogenous DNA stability and its contribution to resistance to Mtz and other DNA damaging agents. RT-PCR of B. fragilis genomic DNA showed that the recA gene was co-transcribed as an operon together with two upstream genes, putatively involved in repairing oxygen damage. A B. fragilis recA mutant was generated using targeted gene inactivation. Fluorescence microscopy using DAPI staining revealed increased numbers of mutant cells with reduced intact double-stranded DNA. Alkaline gel electrophoresis of the recA mutant DNA showed increased amounts of strand breaks under normal growth conditions, and the recA mutant also showed less spontaneous mutagenesis relative to the wild type strain. The recA mutant was sensitive to Mtz, ultraviolet light and hydrogen peroxide. A B. fragilis strain overexpressing the RecA protein exhibited increased resistance to Mtz compared to the wild type. This is the first study to show that overexpression of a DNA repair protein in B. fragilis increases Mtz resistance. This represents a novel drug resistance mechanism in this bacterium. PMID:20435137

Increase in ubiquitin-protein conjugates concomitant with the increase in proteolysis in rat skeletal muscle during starvation and atrophy denervation

NASA Technical Reports Server (NTRS)

Wing, S. S.; Haas, A. L.; Goldberg, A. L.

1995-01-01

The rapid loss of skeletal-muscle protein during starvation and after denervation occurs primarily through increased rates of protein breakdown and activation of a non-lysosomal ATP-dependent proteolytic process. To investigate whether protein flux through the ubiquitin (Ub)-proteasome pathway is enhanced, as was suggested by related studies, we measured, using specific polyclonal antibodies, the levels of Ub-conjugated proteins in normal and atrophying muscles. The content of these critical intermediates had increased 50-250% after food deprivation in the extensor digitorum longus and soleus muscles 2 days after denervation. Like rates of proteolysis, the amount of Ub-protein conjugates and the fraction of Ub conjugated to proteins increased progressively during food deprivation and returned to normal within 1 day of refeeding. During starvation, muscles of adrenalectomized rats failed to increase protein breakdown, and they showed 50% lower levels of Ub-protein conjugates than those of starved control animals. The changes in the pools of Ub-conjugated proteins (the substrates for the 26S proteasome) thus coincided with and can account for the alterations in overall proteolysis. In this pathway, large multiubiquitinated proteins are preferentially degraded, and the Ub-protein conjugates that accumulated in atrophying muscles were of high molecular mass (> 100 kDa). When innervated and denervated gastrocnemius muscles were fractionated, a significant increase in ubiquitinated proteins was found in the myofibrillar fraction, the proteins of which are preferentially degraded on denervation, but not in the soluble fraction. Thus activation of this proteolytic pathway in atrophying muscles probably occurs initially by increasing Ub conjugation to cell proteins. The resulting accumulation of Ub-protein conjugates suggests that their degradation by the 26S proteasome complex subsequently becomes rate-limiting in these catabolic states.

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

EPA Science Inventory

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

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

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

ERIC Educational Resources Information Center

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

2007-01-01

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

Prediction of Enzyme Mutant Activity Using Computational Mutagenesis and Incremental Transduction

PubMed Central

Basit, Nada; Wechsler, Harry

2011-01-01

Wet laboratory mutagenesis to determine enzyme activity changes is expensive and time consuming. This paper expands on standard one-shot learning by proposing an incremental transductive method (T2bRF) for the prediction of enzyme mutant activity during mutagenesis using Delaunay tessellation and 4-body statistical potentials for representation. Incremental learning is in tune with both eScience and actual experimentation, as it accounts for cumulative annotation effects of enzyme mutant activity over time. The experimental results reported, using cross-validation, show that overall the incremental transductive method proposed, using random forest as base classifier, yields better results compared to one-shot learning methods. T2bRF is shown to yield 90% on T4 and LAC (and 86% on HIV-1). This is significantly better than state-of-the-art competing methods, whose performance yield is at 80% or less using the same datasets. PMID:22007208

AXM mutagenesis: an efficient means for the production of libraries for directed evolution of proteins.

PubMed

Holland, Erika G; Buhr, Diane L; Acca, Felicity E; Alderman, Dawn; Bovat, Kristin; Busygina, Valeria; Kay, Brian K; Weiner, Michael P; Kiss, Margaret M

2013-08-30

Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too laborious or expensive to allow high-throughput, parallel processing of multiple antibodies. Here, we describe a scalable approach that enables the generation of libraries with greater than 10(8) clones from a single Escherichia coli transformation. In our method, a mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains at least three phosphorothioate linkages at its 5' end, and treatment of the PCR product with a 5' to 3' exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-stranded DNA fragment. This fragment then serves as a megaprimer to prime DNA synthesis on a uracilated, circular, single-stranded template in a Kunkel-like mutagenesis reaction that biases nucleotide base-changes between the megaprimer and uracilated DNA sequence in favor of the in vitro synthesized megaprimer. This method eliminates the inefficient subcloning steps that are normally required for the construction of affinity maturation libraries from randomly mutagenized antibody genes. Copyright © 2013. Published by Elsevier B.V.

The Effect of α-Mating Factor Secretion Signal Mutations on Recombinant Protein Expression in Pichia pastoris

PubMed Central

Lin-Cereghino, Geoff P.; Stark, Carolyn M.; Kim, Daniel; Chang, Jennifer; Shaheen, Nadia; Poerwanto, Hansel; Agari, Kimiko; Moua, Pachai; Low, Lauren K.; Tran, Namphuong; Huang, Amy D.; Nattestad, Maria; Oshiro, Kristin T.; Chang, John William; Chavan, Archana; Tsai, Jerry W.; Lin-Cereghino, Joan

2013-01-01

The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins are initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future. PMID:23454485

Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system.

PubMed

Chen, Xiugui; Lu, Xuke; Shu, Na; Wang, Shuai; Wang, Junjuan; Wang, Delong; Guo, Lixue; Ye, Wuwei

2017-03-13

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H + -pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6-81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome.

Improvement of lipid production by the oleaginous yeast Rhodosporidium toruloides through UV mutagenesis.

PubMed

Yamada, Ryosuke; Kashihara, Tomomi; Ogino, Hiroyasu

2017-05-01

Oleaginous yeasts are considered a promising alternative lipid source for biodiesel fuel production. In this study, we attempted to improve the lipid productivity of the oleaginous yeast Rhodosporidium toruloides through UV irradiation mutagenesis and selection based on ethanol and H 2 O 2 tolerance or cerulenin, a fatty acid synthetase inhibitor. Glucose consumption, cell growth, and lipid production of mutants were evaluated. The transcription level of genes involved in lipid production was also evaluated in mutants. The ethanol and H 2 O 2 tolerant strain 8766 2-31M and the cerulenin resistant strain 8766 3-11C were generated by UV mutagenesis. The 8766 2-31M mutant showed a higher lipid production rate, and the 8766 3-11C mutant produced a larger amount of lipid and had a higher lipid production rate than the wild type strain. Transcriptional analysis revealed that, similar to the wild type strain, the ACL1 and GND1 genes were expressed at significantly low levels, whereas IDP1 and ME1 were highly expressed. In conclusion, lipid productivity in the oleaginous yeast R. toruloides was successfully improved via UV mutagenesis and selection. The study also identified target genes for improving lipid productivity through gene recombination.

Molecular characterization of a tyrosine-specific protein phosphatase encoded by a stress-responsive gene in Arabidopsis.

PubMed Central

Xu, Q; Fu, H H; Gupta, R; Luan, S

1998-01-01

Protein tyrosine kinases and phosphatases play a vital role in the regulation of cell growth and differentiation in animal systems. However, none of these enzymes has been characterized from higher plants. In this study, we isolated a cDNA encoding a putative protein tyrosine phosphatase (PTPase) from Arabidopsis (referred to as AtPTP1). The expression level of AtPTP1 is highly sensitive to environmental stresses. High-salt conditions increased AtPTP1 mRNA levels, whereas cold treatment rapidly eliminated the AtPTP1 transcript. The recombinant AtPTP1 protein specifically hydrolyzed phosphotyrosine, but not phosphoserine/threonine, in protein substrates. Site-directed mutagenesis defined two highly conserved amino acids, cysteine-265 and aspartate-234, as being essential for the phosphatase activity of the AtPTP1 protein, suggesting a common catalytic mechanism for PTPases from all eukaryotic systems. In summary, we have identified AtPTP1 as a tyrosine-specific protein phosphatase that may function in stress responses of higher plants. PMID:9596642

High-Resolution Functional Mapping of the Venezuelan Equine Encephalitis Virus Genome by Insertional Mutagenesis and Massively Parallel Sequencing

DTIC Science & Technology

2010-10-14

High-Resolution Functional Mapping of the Venezuelan Equine Encephalitis Virus Genome by Insertional Mutagenesis and Massively Parallel Sequencing...Venezuelan equine encephalitis virus (VEEV) genome. We initially used a capillary electrophoresis method to gain insight into the role of the VEEV...Smith JM, Schmaljohn CS (2010) High-Resolution Functional Mapping of the Venezuelan Equine Encephalitis Virus Genome by Insertional Mutagenesis and

Targeted Mutagenesis of Duplicated Genes in Soybean with Zinc-Finger Nucleases1[W][OA

PubMed Central

Curtin, Shaun J.; Zhang, Feng; Sander, Jeffry D.; Haun, William J.; Starker, Colby; Baltes, Nicholas J.; Reyon, Deepak; Dahlborg, Elizabeth J.; Goodwin, Mathew J.; Coffman, Andrew P.; Dobbs, Drena; Joung, J. Keith; Voytas, Daniel F.; Stupar, Robert M.

2011-01-01

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform—a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting DICER-LIKE (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome. PMID:21464476

Rapid mutation of Spirulina platensis by a new mutagenesis system of atmospheric and room temperature plasmas (ARTP) and generation of a mutant library with diverse phenotypes.

PubMed

Fang, Mingyue; Jin, Lihua; Zhang, Chong; Tan, Yinyee; Jiang, Peixia; Ge, Nan; Heping Li; Xing, Xinhui

2013-01-01

In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9(th) subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae.

A divide-and-conquer approach to determine the Pareto frontier for optimization of protein engineering experiments.

PubMed

He, Lu; Friedman, Alan M; Bailey-Kellogg, Chris

2012-03-01

In developing improved protein variants by site-directed mutagenesis or recombination, there are often competing objectives that must be considered in designing an experiment (selecting mutations or breakpoints): stability versus novelty, affinity versus specificity, activity versus immunogenicity, and so forth. Pareto optimal experimental designs make the best trade-offs between competing objectives. Such designs are not "dominated"; that is, no other design is better than a Pareto optimal design for one objective without being worse for another objective. Our goal is to produce all the Pareto optimal designs (the Pareto frontier), to characterize the trade-offs and suggest designs most worth considering, but to avoid explicitly considering the large number of dominated designs. To do so, we develop a divide-and-conquer algorithm, Protein Engineering Pareto FRontier (PEPFR), that hierarchically subdivides the objective space, using appropriate dynamic programming or integer programming methods to optimize designs in different regions. This divide-and-conquer approach is efficient in that the number of divisions (and thus calls to the optimizer) is directly proportional to the number of Pareto optimal designs. We demonstrate PEPFR with three protein engineering case studies: site-directed recombination for stability and diversity via dynamic programming, site-directed mutagenesis of interacting proteins for affinity and specificity via integer programming, and site-directed mutagenesis of a therapeutic protein for activity and immunogenicity via integer programming. We show that PEPFR is able to effectively produce all the Pareto optimal designs, discovering many more designs than previous methods. The characterization of the Pareto frontier provides additional insights into the local stability of design choices as well as global trends leading to trade-offs between competing criteria. Copyright © 2011 Wiley Periodicals, Inc.

Post-Weaning Protein Malnutrition Increases Blood Pressure and Induces Endothelial Dysfunctions in Rats

PubMed Central

Siman, Fabiana D. M.; Silveira, Edna A.; Meira, Eduardo F.; da Costa, Carlos P.; Vassallo, Dalton V.; Padilha, Alessandra S.

2012-01-01

Malnutrition during critical periods in early life may increase the subsequent risk of hypertension and metabolic diseases in adulthood, but the underlying mechanisms are still unclear. We aimed to evaluate the effects of post-weaning protein malnutrition on blood pressure and vascular reactivity in aortic rings (conductance artery) and isolated-perfused tail arteries (resistance artery) from control (fed with Labina®) and post-weaning protein malnutrition rats (offspring that received a diet with low protein content for three months). Systolic and diastolic blood pressure and heart rate increased in the post-weaning protein malnutrition rats. In the aortic rings, reactivity to phenylephrine (10−10–3.10−4 M) was similar in both groups. Endothelium removal or L-NAME (10−4 M) incubation increased the response to phenylephrine, but the L-NAME effect was greater in the aortic rings from the post-weaning protein malnutrition rats. The protein expression of the endothelial nitric oxide isoform increased in the aortic rings from the post-weaning protein malnutrition rats. Incubation with apocynin (0.3 mM) reduced the response to phenylephrine in both groups, but this effect was higher in the post-weaning protein malnutrition rats, suggesting an increase of superoxide anion release. In the tail artery of the post-weaning protein malnutrition rats, the vascular reactivity to phenylephrine (0.001–300 µg) and the relaxation to acetylcholine (10−10–10−3 M) were increased. Post-weaning protein malnutrition increases blood pressure and induces vascular dysfunction. Although the vascular reactivity in the aortic rings did not change, an increase in superoxide anion and nitric oxide was observed in the post-weaning protein malnutrition rats. However, in the resistance arteries, the increased vascular reactivity may be a potential mechanism underlying the increased blood pressure observed in this model. PMID:22529948

Hydrophobic interaction chromatography in dual salt system increases protein binding capacity.

PubMed

Senczuk, Anna M; Klinke, Ralph; Arakawa, Tsutomu; Vedantham, Ganesh; Yigzaw, Yinges

2009-08-01

Hydrophobic interaction chromatography (HIC) uses weakly hydrophobic resins and requires a salting-out salt to promote protein-resin interaction. The salting-out effects increase with protein and salt concentration. Dynamic binding capacity (DBC) is dependent on the binding constant, as well as on the flow characteristics during sample loading. DBC increases with the salt concentration but decreases with increasing flow rate. Dynamic and operational binding capacity have a major raw material cost/processing time impact on commercial scale production of monoclonal antibodies. In order to maximize DBC the highest salt concentration without causing precipitation is used. We report here a novel method to maintain protein solubility while increasing the DBC by using a combination of two salting-out salts (referred to as dual salt). In a series of experiments, we explored the dynamic capacity of a HIC resin (TosoBioscience Butyl 650M) with combinations of salts. Using a model antibody, we developed a system allowing us to increase the dynamic capacity up to twofold using the dual salt system over traditional, single salt system. We also investigated the application of this novel approach to several other proteins and salt combinations, and noted a similar protein solubility and DBC increase. The observed increase in DBC in the dual salt system was maintained at different linear flow rates and did not impact selectivity.

Genome-wide transposon mutagenesis in pathogenic Leptospira species.

PubMed

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

2009-02-01

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

Identification of virulence determinants for endocarditis in Streptococcus sanguinis by signature-tagged mutagenesis.

PubMed

Paik, Sehmi; Senty, Lauren; Das, Sankar; Noe, Jody C; Munro, Cindy L; Kitten, Todd

2005-09-01

Streptococcus sanguinis is a gram-positive, facultative anaerobe and a normal inhabitant of the human oral cavity. It is also one of the most common agents of infective endocarditis, a serious endovascular infection. To identify virulence factors for infective endocarditis, signature-tagged mutagenesis (STM) was applied to the SK36 strain of S. sanguinis, whose genome is being sequenced. STM allows the large-scale creation, in vivo screening, and recovery of a series of mutants with altered virulence. Screening of 800 mutants by STM identified 38 putative avirulent and 5 putative hypervirulent mutants. Subsequent molecular analysis of a subset of these mutants identified genes encoding undecaprenol kinase, homoserine kinase, anaerobic ribonucleotide reductase, adenylosuccinate lyase, and a hypothetical protein. Virulence reductions ranging from 2-to 150-fold were confirmed by competitive index assays. One putatively hypervirulent strain with a transposon insertion in an intergenic region was identified, though increased virulence was not confirmed in competitive index assays. All mutants grew comparably to SK36 in aerobic broth culture except for the homoserine kinase mutant. Growth of this mutant was restored by the addition of threonine to the medium. Mutants containing an insertion or in-frame deletion in the anaerobic ribonucleotide reductase gene failed to grow under strictly anaerobic conditions. The results suggest that housekeeping functions such as cell wall synthesis, amino acid and nucleic acid synthesis, and the ability to survive under anaerobic conditions are important virulence factors in S. sanguinis endocarditis.

Foods with increased protein content: A qualitative study on European consumer preferences and perceptions.

PubMed

Banovic, Marija; Arvola, Anne; Pennanen, Kyösti; Duta, Denisa E; Brückner-Gühmann, Monika; Lähteenmäki, Liisa; Grunert, Klaus G

2018-06-01

Foods with increased protein content have rapidly become one of the fastest-growing product categories targeting image- and health-focused consumers. However, it is not clear whether consumers really understand the difference between 'inherently rich in protein' and 'artificially increased protein'. This study used a qualitative focus group approach to investigate the consumer preferences and perceptions of foods with increased protein content among mixed-age and older population in four European countries. In total fifty-two participants were involved in the study. Understanding of the concept of foods with 'increased protein' content was limited. Both older and mixed-age participants could not differentiate between natural sources of protein and foods with increased protein content, no matter whether foods with animal or plant proteins were mentioned. Older participants expressed more scepticism towards foods with increased protein content than mixed-age participants. The combination of protein type and food carrier closer to conventional foods received more acceptance among both older and mixed-age participants. Future use and acceptance of foods with increased protein content will depend on the extent to which consumer concerns about incorporating additional protein into a diet can be responded. Copyright © 2018 Elsevier Ltd. All rights reserved.

Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci.

PubMed

Canver, Matthew C; Lessard, Samuel; Pinello, Luca; Wu, Yuxuan; Ilboudo, Yann; Stern, Emily N; Needleman, Austen J; Galactéros, Frédéric; Brugnara, Carlo; Kutlar, Abdullah; McKenzie, Colin; Reid, Marvin; Chen, Diane D; Das, Partha Pratim; A Cole, Mitchel; Zeng, Jing; Kurita, Ryo; Nakamura, Yukio; Yuan, Guo-Cheng; Lettre, Guillaume; Bauer, Daniel E; Orkin, Stuart H

2017-04-01

Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, which is associated with red-blood-cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false-positive regions, thus emphasizing the importance of off-target analysis in the design of saturating-mutagenesis experiments. Together, these data establish a widely applicable high-throughput and high-resolution methodology to identify minimal functional sequences within large disease- and trait-associated regions.

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

PubMed Central

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

2013-01-01

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

Structural Analysis and Deletion Mutagenesis Define Regions of QUIVER/SLEEPLESS that Are Responsible for Interactions with Shaker-Type Potassium Channels and Nicotinic Acetylcholine Receptors

PubMed Central

Wu, Meilin; Liu, Clifford Z.; Joiner, William J.

2016-01-01

Ly6 proteins are endogenous prototoxins found in most animals. They show striking structural and functional parallels to snake α-neurotoxins, including regulation of ion channels and cholinergic signaling. However, the structural contributions of Ly6 proteins to regulation of effector molecules is poorly understood. This question is particularly relevant to the Ly6 protein QUIVER/SLEEPLESS (QVR/SSS), which has previously been shown to suppress excitability and synaptic transmission by upregulating potassium (K) channels and downregulating nicotinic acetylcholine receptors (nAChRs) in wake-promoting neurons to facilitate sleep in Drosophila. Using deletion mutagenesis, co-immunoprecipitations, ion flux assays, surface labeling and confocal microscopy, we demonstrate that only loop 2 is required for many of the previously described properties of SSS in transfected cells, including interactions with K channels and nAChRs. Collectively our data suggest that QVR/SSS, and by extension perhaps other Ly6 proteins, target effector molecules using limited protein motifs. Mapping these motifs may be useful in rational design of drugs that mimic or suppress Ly6-effector interactions to modulate nervous system function. PMID:26828958

COPS5 (Jab1) protein increases β site processing of amyloid precursor protein and amyloid β peptide generation by stabilizing RanBP9 protein levels.

PubMed

Wang, Hongjie; Dey, Debleena; Carrera, Ivan; Minond, Dmitriy; Bianchi, Elisabetta; Xu, Shaohua; Lakshmana, Madepalli K

2013-09-13

Increased processing of amyloid precursor protein (APP) and accumulation of neurotoxic amyloid β peptide (Aβ) in the brain is central to the pathogenesis of Alzheimer's disease (AD). Therefore, the identification of molecules that regulate Aβ generation is crucial for future therapeutic approaches for AD. We demonstrated previously that RanBP9 regulates Aβ generation in a number of cell lines and primary neuronal cultures by forming tripartite protein complexes with APP, low-density lipoprotein-related protein, and BACE1, consequently leading to increased amyloid plaque burden in the brain. RanBP9 is a scaffold protein that exists and functions in multiprotein complexes. To identify other proteins that may bind RanBP9 and regulate Aβ levels, we used a two-hybrid analysis against a human brain cDNA library and identified COPS5 as a novel RanBP9-interacting protein. This interaction was confirmed by coimmunoprecipitation experiments in both neuronal and non-neuronal cells and mouse brain. Colocalization of COPS5 and RanBP9 in the same subcellular compartments further supported the interaction of both proteins. Furthermore, like RanBP9, COPS5 robustly increased Aβ generation, followed by increased soluble APP-β (sAPP-β) and decreased soluble-APP-α (sAPP-α) levels. Most importantly, down-regulation of COPS5 by siRNAs reduced Aβ generation, implying that endogenous COPS5 regulates Aβ generation. Finally, COPS5 levels were increased significantly in AD brains and APΔE9 transgenic mice, and overexpression of COPS5 strongly increased RanBP9 protein levels by increasing its half-life. Taken together, these results suggest that COPS5 increases Aβ generation by increasing RanBP9 levels. Thus, COPS5 is a novel RanBP9-binding protein that increases APP processing and Aβ generation by stabilizing RanBP9 protein levels.

Sex differences in snack food reinforcement in response to increasing dietary protein

USDA-ARS?s Scientific Manuscript database

BRACKGROUND: Protein is posited to play a dynamic role in energy balance and reward-driven eating behavior. However, little is known about the effect of increasing protein intake on snack food reinforcement. OBJECTIVE: We sought to determine the extent to which increasing dietary protein changes th...

Schematic representation of residue-based protein context-dependent data: an application to transmembrane proteins.

PubMed

Campagne, F; Weinstein, H

1999-01-01

An algorithmic method for drawing residue-based schematic diagrams of proteins on a 2D page is presented and illustrated. The method allows the creation of rendering engines dedicated to a given family of sequences, or fold. The initial implementation provides an engine that can produce a 2D diagram representing secondary structure for any transmembrane protein sequence. We present the details of the strategy for automating the drawing of these diagrams. The most important part of this strategy is the development of an algorithm for laying out residues of a loop that connects to arbitrary points of a 2D plane. As implemented, this algorithm is suitable for real-time modification of the loop layout. This work is of interest for the representation and analysis of data from (1) protein databases, (2) mutagenesis results, or (3) various kinds of protein context-dependent annotations or data.

Virus-Like Particles Derived from HIV-1 for Delivery of Nuclear Proteins: Improvement of Production and Activity by Protein Engineering.

PubMed

Robert, Marc-André; Lytvyn, Viktoria; Deforet, Francis; Gilbert, Rénald; Gaillet, Bruno

2017-01-01

Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4). The sizes of the VLPs were 120-150 nm in diameter and they were resistant to freeze/thaw cycles. Protein delivery by the VLPs reached up to 100% efficacy in human cells and was well tolerated. Gag-cTA triggered up to 1100-fold gene activation of the reporter gene in comparison to the negative control. Protein engineering was required to detect Gag-KLF4 activity. Thus, insertion of the VP16 transactivation domain increased the activity of the VLPs by eightfold. An additional 2.4-fold enhancement was obtained by inserting nuclear export signal. In conclusion, our platform produced VLPs capable of efficient protein transfer, and it was shown that protein engineering can be used to improve the activity of the delivered proteins as well as VLP production.

Can enzyme engineering benefit from the modulation of protein motions? Lessons learned from NMR relaxation dispersion experiments.

PubMed

Doucet, Nicolas

2011-04-01

Despite impressive progress in protein engineering and design, our ability to create new and efficient enzyme activities remains a laborious and time-consuming endeavor. In the past few years, intricate combinations of rational mutagenesis, directed evolution and computational methods have paved the way to exciting engineering examples and are now offering a new perspective on the structural requirements of enzyme activity. However, these structure-function analyses are usually guided by the time-averaged static models offered by enzyme crystal structures, which often fail to describe the functionally relevant 'invisible states' adopted by proteins in space and time. To alleviate such limitations, NMR relaxation dispersion experiments coupled to mutagenesis studies have recently been applied to the study of enzyme catalysis, effectively complementing 'structure-function' analyses with 'flexibility-function' investigations. In addition to offering quantitative, site-specific information to help characterize residue motion, these NMR methods are now being applied to enzyme engineering purposes, providing a powerful tool to help characterize the effects of controlling long-range networks of flexible residues affecting enzyme function. Recent advancements in this emerging field are presented here, with particular attention to mutagenesis reports highlighting the relevance of NMR relaxation dispersion tools in enzyme engineering.

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

PubMed Central

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

1999-01-01

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

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins.

PubMed

Yao, Xin-Qiu; Malik, Rabia U; Griggs, Nicholas W; Skjærven, Lars; Traynor, John R; Sivaramakrishnan, Sivaraj; Grant, Barry J

2016-02-26

G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gαi. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system

PubMed Central

Chen, Xiugui; Lu, Xuke; Shu, Na; Wang, Shuai; Wang, Junjuan; Wang, Delong; Guo, Lixue; Ye, Wuwei

2017-01-01

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H+-pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6–81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome. PMID:28287154

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

PubMed

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

2017-01-01

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

Bryn Bridges and mutagenesis: exploring the intellectual space.

PubMed

Walker, G C

2001-02-25

The products of the SOS-regulated umuDC genes are required for most UV and chemical mutagenesis in Escherichia coli. Recently it has been recognized that UmuC is the founding member of a superfamily of novel DNA polymerases found in all three kingdoms of life. Key findings leading to these insights are reviewed, placing a particular emphasis on contributions made by Bryn Bridges and on his interest in the importance of interactions between the umuDC gene products and the replicative DNA polymerase.

A protein functional leap: how a single mutation reverses the function of the transcription regulator TetR.

PubMed

Resch, Marcus; Striegl, Harald; Henssler, Eva Maria; Sevvana, Madhumati; Egerer-Sieber, Claudia; Schiltz, Emile; Hillen, Wolfgang; Muller, Yves A

2008-08-01

Today's proteome is the result of innumerous gene duplication, mutagenesis, drift and selection processes. Whereas random mutagenesis introduces predominantly only gradual changes in protein function, a case can be made that an abrupt switch in function caused by single amino acid substitutions will not only considerably further evolution but might constitute a prerequisite for the appearance of novel functionalities for which no promiscuous protein intermediates can be envisaged. Recently, tetracycline repressor (TetR) variants were identified in which binding of tetracycline triggers the repressor to associate with and not to dissociate from the operator DNA as in wild-type TetR. We investigated the origin of this activity reversal by limited proteolysis, CD spectroscopy and X-ray crystallography. We show that the TetR mutant Leu17Gly switches its function via a disorder-order mechanism that differs completely from the allosteric mechanism of wild-type TetR. Our study emphasizes how single point mutations can engender unexpected leaps in protein function thus enabling the appearance of new functionalities in proteins without the need for promiscuous intermediates.

TP53 mutations in squamous-cell carcinomas of the conjunctiva: evidence for UV-induced mutagenesis.

PubMed

Ateenyi-Agaba, Charles; Dai, Min; Le Calvez, Florence; Katongole-Mbidde, Edward; Smet, Anouk; Tommasino, Massimo; Franceschi, Silvia; Hainaut, Pierre; Weiderpass, Elisabete

2004-09-01

Squamous cell carcinoma of the conjunctiva is associated with sun exposure and often occurs in HIV-positive individuals. We have analysed TP53 mutations in 21 cases of squamous cell carcinoma and 22 controls with benign conjunctival lesions from a region (Uganda, Africa) with a high prevalence of heavy sun exposure and HIV infection. TP53 mutations were detected in 11 cases (52%) and 3 controls (14%). Seven of the mutations (6 in cases and 1 in controls) were CC-->TT transitions, a molecular signature of mutagenesis by solar UV rays. A similar prevalence (56%) of TP53 mutations was found in 18 squamous cell carcinoma cases positive for epidermodysplasia verruciformis human papillomavirus types. The prevalence of CC-->TT transitions reported here is the highest observed in any cancer type and matches that of skin cancers in subjects with xeroderma pigmentosum, an inherited disease with hypersensitivity to UV damage. These results confirm at the molecular level the causal role of solar UV rays in the aetiology of squamous cell carcinoma of the conjunctiva and suggest that infection with epidermodysplasia verruciformis types of human papillomavirus may act as a cofactor to increase the sensitivity of conjunctiva cells to UV-induced mutagenesis.

Advanced protein formulations

PubMed Central

Wang, Wei

2015-01-01

It is well recognized that protein product development is far more challenging than that for small-molecule drugs. The major challenges include inherent sensitivity to different types of stresses during the drug product manufacturing process, high rate of physical and chemical degradation during long-term storage, and enhanced aggregation and/or viscosity at high protein concentrations. In the past decade, many novel formulation concepts and technologies have been or are being developed to address these product development challenges for proteins. These concepts and technologies include use of uncommon/combination of formulation stabilizers, conjugation or fusion with potential stabilizers, site-specific mutagenesis, and preparation of nontraditional types of dosage forms—semiaqueous solutions, nonfreeze-dried solid formulations, suspensions, and other emerging concepts. No one technology appears to be mature, ideal, and/or adequate to address all the challenges. These gaps will likely remain in the foreseeable future and need significant efforts for ultimate resolution. PMID:25858529

Ingestion of Wheat Protein Increases In Vivo Muscle Protein Synthesis Rates in Healthy Older Men in a Randomized Trial.

PubMed

Gorissen, Stefan Hm; Horstman, Astrid Mh; Franssen, Rinske; Crombag, Julie Jr; Langer, Henning; Bierau, Jörgen; Respondek, Frederique; van Loon, Luc Jc

2016-09-01

Muscle mass maintenance is largely regulated by basal muscle protein synthesis and the capacity to stimulate muscle protein synthesis after food intake. The postprandial muscle protein synthetic response is modulated by the amount, source, and type of protein consumed. It has been suggested that plant-based proteins are less potent in stimulating postprandial muscle protein synthesis than animal-derived proteins. However, few data support this contention. We aimed to assess postprandial plasma amino acid concentrations and muscle protein synthesis rates after the ingestion of a substantial 35-g bolus of wheat protein hydrolysate compared with casein and whey protein. Sixty healthy older men [mean ± SEM age: 71 ± 1 y; body mass index (in kg/m(2)): 25.3 ± 0.3] received a primed continuous infusion of l-[ring-(13)C6]-phenylalanine and ingested 35 g wheat protein (n = 12), 35 g wheat protein hydrolysate (WPH-35; n = 12), 35 g micellar casein (MCas-35; n = 12), 35 g whey protein (Whey-35; n = 12), or 60 g wheat protein hydrolysate (WPH-60; n = 12). Plasma and muscle samples were collected at regular intervals. The postprandial increase in plasma essential amino acid concentrations was greater after ingesting Whey-35 (2.23 ± 0.07 mM) than after MCas-35 (1.53 ± 0.08 mM) and WPH-35 (1.50 ± 0.04 mM) (P < 0.01). Myofibrillar protein synthesis rates increased after ingesting MCas-35 (P < 0.01) and were higher after ingesting MCas-35 (0.050% ± 0.005%/h) than after WPH-35 (0.032% ± 0.004%/h) (P = 0.03). The postprandial increase in plasma leucine concentrations was greater after ingesting Whey-35 than after WPH-60 (peak value: 580 ± 18 compared with 378 ± 10 μM, respectively; P < 0.01), despite similar leucine contents (4.4 g leucine). Nevertheless, the ingestion of WPH-60 increased myofibrillar protein synthesis rates above basal rates (0.049% ± 0.007%/h; P = 0.02). The myofibrillar protein synthetic response to the ingestion of 35 g casein is greater than after an

A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning.

PubMed

Wan, Haisu; Li, Yongwen; Fan, Yu; Meng, Fanrong; Chen, Chen; Zhou, Qinghua

2012-01-15

Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer-template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function. Copyright © 2011 Elsevier Inc. All rights reserved.

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

PubMed

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

2016-07-01

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

HIV-1 Protease Function and Structure Studies with the Simplicial Neighborhood Analysis of Protein Packing (SNAPP) Method

PubMed Central

Zhang, Shuxing; Kaplan, Andrew H.; Tropsha, Alexander

2009-01-01

The Simplicial Neighborhood Analysis of Protein Packing (SNAPP) method was used to predict the effect of mutagenesis on the enzymatic activity of the HIV-1 protease (HIVP). SNAPP relies on a four-body statistical scoring function derived from the analysis of spatially nearest neighbor residue compositional preferences in a diverse and representative subset of protein structures from the Protein Data Bank. The method was applied to the analysis of HIVP mutants with residue substitutions in the hydrophobic core as well as at the interface between the two protease monomers. Both wild type and tethered structures were employed in the calculations. We obtained a strong correlation, with R2 as high as 0.96, between ΔSNAPP score (i.e., the difference in SNAPP scores between wild type and mutant proteins) and the protease catalytic activity for tethered structures. A weaker but significant correlation was also obtained for non-tethered structures as well. Our analysis identified residues both in the hydrophobic core and at the dimeric interface (DI) that are very important for the protease function. This study demonstrates a potential utility of the SNAPP method for rational design of mutagenesis studies and protein engineering. PMID:18498108

Mutagenesis of the La Crosse Virus glycoprotein supports a role for Gc (1066-1087) as the fusion peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Plassmeyer, Matthew L.; Graduate Group Molecular and Cell Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058; Soldan, Samantha S.

The La Crosse Virus (LACV) M segment encodes two glycoproteins (Gn and Gc), and plays a critical role in the neuropathogenesis of LACV infection as the primary determinant of neuroinvasion. A recent study from our group demonstrated that the region comprising the membrane proximal two-thirds of Gc, amino acids 860-1442, is critical in mediating LACV fusion and entry. Furthermore, computational analysis identified structural similarities between a portion of this region, amino acids 970-1350, and the E1 fusion protein of two alphaviruses: Sindbis virus and Semliki Forrest virus (SFV). Within the region 970-1350, a 22-amino-acid hydrophobic segment (1066-1087) is predicted tomore » correlate structurally with the fusion peptides of class II fusion proteins. We performed site-directed mutagenesis of key amino acids in this 22-amino acid segment and determined the functional consequences of these mutations on fusion and entry. Several mutations within this hydrophobic domain affected glycoprotein expression to some extent, but all mutations either shifted the pH threshold of fusion below that of the wild-type protein, reduced fusion efficiency, or abrogated cell-to-cell fusion and pseudotype entry altogether. These results, coupled with the aforementioned computational modeling, suggest that the LACV Gc functions as a class II fusion protein and support a role for the region Gc 1066-1087 as a fusion peptide.« less

Rapid Mutation of Spirulina platensis by a New Mutagenesis System of Atmospheric and Room Temperature Plasmas (ARTP) and Generation of a Mutant Library with Diverse Phenotypes

PubMed Central

Zhang, Chong; Tan, Yinyee; Jiang, Peixia; Ge, Nan; Heping Li; Xing, Xinhui

2013-01-01

In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae. PMID:24319517

Chiral mutagenesis of insulin. Contribution of the B20-B23 beta-turn to activity and stability.

PubMed

Nakagawa, Satoe H; Hua, Qing-xin; Hu, Shi-Quan; Jia, Wenhua; Wang, Shuhua; Katsoyannis, Panayotis G; Weiss, Michael A

2006-08-04

Insulin contains a beta-turn (residues B20-B23) interposed between two receptor-binding elements, the central alpha-helix of the B chain (B9-B19) and its C-terminal beta-strand (B24-B28). The turn contains conserved glycines at B20 and B23. Although insulin exhibits marked conformational variability among crystal forms, these glycines consistently maintain positive phi dihedral angles within a classic type-I beta-turn. Because the Ramachandran conformations of GlyB20 and GlyB23 are ordinarily forbidden to L-amino acids, turn architecture may contribute to structure or function. Here, we employ "chiral mutagenesis," comparison of corresponding D- and L-Ala substitutions, to investigate this turn. Control substitutions are introduced at GluB21, a neighboring residue exhibiting a conventional (negative) phi angle. The D- and L-Ala substitutions at B23 are associated with a marked stereospecific difference in activity. Whereas the D-AlaB23 analog retains native activity, the L analog exhibits a 20-fold decrease in receptor binding. By contrast, D- and L-AlaB20 analogs each exhibit high activity. Stereospecific differences between the thermodynamic stabilities of the analogs are nonetheless more pronounced at B20 (delta deltaG(u) 2.0 kcal/mole) than at B23 (delta deltaG(u) 0.7 kcal/mole). Control substitutions at B21 are well tolerated without significant stereospecificity. Chiral mutagenesis thus defines the complementary contributions of these conserved glycines to protein stability (GlyB20) or receptor recognition (GlyB23).

Targeted mutagenesis of the psbE and psbF genes blocks photosynthetic electron transport: evidence for a functional role of cytochrome b559 in photosystem II.

PubMed Central

Pakrasi, H B; Williams, J G; Arntzen, C J

1988-01-01

The genes encoding the two subunits (alpha and beta) of the cytochrome b559 (cyt b559) protein, psbE and psbF, were cloned from the unicellular, transformable cyanobacterium, Synechocystis 6803. Cyt b559, an intrinsic membrane protein, is a component of photosystem II, a membrane-protein complex that catalyzes photosynthetic oxygen evolution. However, the role of cyt b559 in photosynthetic electron transport is yet to be determined. A high degree of homology was found between the cyanobacterial and green plant chloroplastidic psbE and psbE genes and in the amino acid sequences of their corresponding protein products. Cartridge mutagenesis techniques were used to generate a deletion mutant of Synechocystis 6803 in which the psbE and psbF genes were replaced by a kanamycin-resistance gene cartridge. Physiological analyses indicated that the PSII complexes of the mutant were inactivated. We conclude that cyt b559 is an essential component of PSII. Images PMID:3130246

Synergistic interaction between excess hepatic iron and alcohol ingestion in hepatic mutagenesis.

PubMed

Asare, George A; Bronz, Michelle; Naidoo, Vivash; Kew, Michael C

2008-12-05

Hereditary hemochromatosis (HH) and dietary iron overload are the main iron-loading diseases. Fibrosis, cirrhosis and hepatocellular carcinoma (HCC) are complications to HH and dietary iron overload possibly influenced by co-factors. Alcohol may be one such factor. The aim therefore was to determine the extent of synergistic interaction between free hepatic iron and alcohol, complicating dietary iron overload in HCC pathogenesis. Four groups of 20 Wistar albino rats were used: group 1 (C) was fed the chow diet; group 2 (Fe) was supplemented with 0.75% ferrocene iron; group 3 (Fe+Al), 0.75% iron and 7% ethanol; and group 4, 7% ethanol (Al) for 12 months. Iron profile, superoxide/nitrite free radicals, lipid peroxidation (LPO)/8-isoprostane (8-IP), 8-hydroxydeoxyguanosine (8-OHdG), oxidative lipid/DNA damage immunohistochemistry, transaminases (AST/ALT) and Ames mutagenesis tests were performed. Significant differences were observed in the Fe+Al group for LPO, 8-IP, AST and ALT (p<0.001, 0.001, 0.001 and 0.001, respectively) compared to other groups. A three-fold synergistic interaction was observed for the same parameters. Furthermore, significant differences of p<0.05 and 0.001 were observed for 8-OHdG and mutagenesis, respectively, with an additive synergy in the Fe+Al group. ALT/8-OHdG and ALT/mutagenesis correlated positively (p<0.04 and 0.008, respectively). The immunohistochemistry revealed iron/alcohol multiplicative synergism with hydroxyl radical involvement. Mutagenic effects of iron and alcohol are synergistically multiplicative implicating hydroxyl free radicals in hepatocarcingenesis.

Simulation and estimation of gene number in a biological pathway using almost complete saturation mutagenesis screening of haploid mouse cells.

PubMed

Tokunaga, Masahiro; Kokubu, Chikara; Maeda, Yusuke; Sese, Jun; Horie, Kyoji; Sugimoto, Nakaba; Kinoshita, Taroh; Yusa, Kosuke; Takeda, Junji

2014-11-24

Genome-wide saturation mutagenesis and subsequent phenotype-driven screening has been central to a comprehensive understanding of complex biological processes in classical model organisms such as flies, nematodes, and plants. The degree of "saturation" (i.e., the fraction of possible target genes identified) has been shown to be a critical parameter in determining all relevant genes involved in a biological function, without prior knowledge of their products. In mammalian model systems, however, the relatively large scale and labor intensity of experiments have hampered the achievement of actual saturation mutagenesis, especially for recessive traits that require biallelic mutations to manifest detectable phenotypes. By exploiting the recently established haploid mouse embryonic stem cells (ESCs), we present an implementation of almost complete saturation mutagenesis in a mammalian system. The haploid ESCs were mutagenized with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and processed for the screening of mutants defective in various steps of the glycosylphosphatidylinositol-anchor biosynthetic pathway. The resulting 114 independent mutant clones were characterized by a functional complementation assay, and were shown to be defective in any of 20 genes among all 22 known genes essential for this well-characterized pathway. Ten mutants were further validated by whole-exome sequencing. The predominant generation of single-nucleotide substitutions by ENU resulted in a gene mutation rate proportional to the length of the coding sequence, which facilitated the experimental design of saturation mutagenesis screening with the aid of computational simulation. Our study enables mammalian saturation mutagenesis to become a realistic proposition. Computational simulation, combined with a pilot mutagenesis experiment, could serve as a tool for the estimation of the number of genes essential for biological processes such as drug target pathways when a positive selection of

Inflating bacterial cells by increased protein synthesis

PubMed Central

Basan, Markus; Zhu, Manlu; Dai, Xiongfeng; Warren, Mya; Sévin, Daniel; Wang, Yi-Ping; Hwa, Terence

2015-01-01

Understanding how the homeostasis of cellular size and composition is accomplished by different organisms is an outstanding challenge in biology. For exponentially growing Escherichia coli cells, it is long known that the size of cells exhibits a strong positive relation with their growth rates in different nutrient conditions. Here, we characterized cell sizes in a set of orthogonal growth limitations. We report that cell size and mass exhibit positive or negative dependences with growth rate depending on the growth limitation applied. In particular, synthesizing large amounts of “useless” proteins led to an inversion of the canonical, positive relation, with slow growing cells enlarged 7- to 8-fold compared to cells growing at similar rates under nutrient limitation. Strikingly, this increase in cell size was accompanied by a 3- to 4-fold increase in cellular DNA content at slow growth, reaching up to an amount equivalent to ∼8 chromosomes per cell. Despite drastic changes in cell mass and macromolecular composition, cellular dry mass density remained constant. Our findings reveal an important role of protein synthesis in cell division control. PMID:26519362

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

PubMed Central

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

1990-01-01

Azotobacter vinelandii ferredoxin I is a small protein that contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. Recently the x-ray crystal structure has been redetermined and the fdxA gene, which encodes the protein, has been cloned and sequenced. Here we report the site-directed mutation of Cys-20, which is a ligand of the [4Fe-4S] cluster in the native protein, to alanine and the characterization of the protein product by x-ray crystallographic and spectroscopic methods. The data show that the mutant protein again contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. The new [4Fe-4S] cluster obtains its fourth ligand from Cys-24, a free cysteine in the native structure. The formation of this [4Fe-4S] cluster drives rearrangement of the protein structure. PMID:2153958

Dissecting binding of a β-barrel membrane protein by phage display.

PubMed

Meneghini, Luz M; Tripathi, Sarvind; Woodworth, Marcus A; Majumdar, Sudipta; Poulos, Thomas L; Weiss, Gregory A

2017-07-25

Membrane proteins (MPs) constitute a third of all proteomes, and contribute to a myriad of cellular functions including intercellular communication, nutrient transport and energy generation. For example, TonB-dependent transporters (TBDTs) in the outer membrane of Gram-negative bacteria play an essential role transporting iron and other nutrients into the bacterial cell. The inherently hydrophobic surfaces of MPs complicates protein expression, purification, and characterization. Thus, dissecting the functional contributions of individual amino acids or structural features through mutagenesis can be a challenging ordeal. Here, we apply a new approach for the expedited protein characterization of the TBDT ShuA from Shigella dysenteriae, and elucidate the protein's initial steps during heme-uptake. ShuA variants were displayed on the surface of an M13 bacteriophage as fusions to the P8 coat protein. Each ShuA variant was analyzed for its ability to display on the bacteriophage surface, and functionally bind to hemoglobin. This technique streamlines isolation of stable MP variants for rapid characterization of binding to various ligands. Site-directed mutagenesis studies targeting each extracellular loop region of ShuA demonstrate no specific extracellular loop is required for hemoglobin binding. Instead two residues, His420 and His86 mediate this interaction. The results identify a loop susceptible to antibody binding, and also a small molecule motif capable of disrupting ShuA from S. dysenteriae. The approach is generalizable to the dissection of other phage-displayed TBDTs and MPs.

G protein-coupled odorant receptors: From sequence to structure.

PubMed

de March, Claire A; Kim, Soo-Kyung; Antonczak, Serge; Goddard, William A; Golebiowski, Jérôme

2015-09-01

Odorant receptors (ORs) are the largest subfamily within class A G protein-coupled receptors (GPCRs). No experimental structural data of any OR is available to date and atomic-level insights are likely to be obtained by means of molecular modeling. In this article, we critically align sequences of ORs with those GPCRs for which a structure is available. Here, an alignment consistent with available site-directed mutagenesis data on various ORs is proposed. Using this alignment, the choice of the template is deemed rather minor for identifying residues that constitute the wall of the binding cavity or those involved in G protein recognition. © 2015 The Protein Society.

Oxysterol-binding protein-related protein (ORP) 9 is a PDK-2 substrate and regulates Akt phosphorylation.

PubMed

Lessmann, Eva; Ngo, Mike; Leitges, Michael; Minguet, Susana; Ridgway, Neale D; Huber, Michael

2007-02-01

The oxysterol-binding protein and oxysterol-binding protein-related protein family has been implicated in lipid transport and metabolism, vesicle trafficking and cell signaling. While investigating the phosphorylation of Akt/protein kinase B in stimulated bone marrow-derived mast cells, we observed that a monoclonal antibody directed against phospho-S473 Akt cross-reacted with oxysterol-binding protein-related protein 9 (ORP9). Further analysis revealed that mast cells exclusively express ORP9S, an N-terminal truncated version of full-length ORP9L. A PDK-2 consensus phosphorylation site in ORP9L and OPR9S at S287 (VPEFS(287)Y) was confirmed by site-directed mutagenesis. In contrast to Akt, increased phosphorylation of ORP9S S287 in stimulated mast cells was independent of phosphatidylinositol 3-kinase but sensitive to inhibition of conventional PKC isotypes. PKC-beta dependence was confirmed by lack of ORP9S phosphorylation at S287 in PKC-beta-deficient, but not PKC-alpha-deficient, mast cells. Moreover, co-immunoprecipitation of PKC-beta and ORP9S, and in vitro phosphorylation of ORP9S in this complex, argued for direct phosphorylation of ORP9S by PKC-beta, introducing ORP9S as a novel PKC-beta substrate. Akt was also detected in a PKC-beta/ORP9S immune complex and phosphorylation of Akt on S473 was delayed in PKC-deficient mast cells. In HEK293 cells, RNAi experiments showed that depletion of ORP9L increased Akt S473 phosphorylation 3-fold without affecting T308 phosphorylation in the activation loop. Furthermore, mammalian target of rapamycin was implicated in ORP9L phosphorylation in HEK293 cells. These studies identify ORP9 as a PDK-2 substrate and negative regulator of Akt phosphorylation at the PDK-2 site.

NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells

NASA Technical Reports Server (NTRS)

Zhang, Ying; Lim, Chang U K.; Williams, Eli S.; Zhou, Junqing; Zhang, Qinming; Fox, Michael H.; Bailey, Susan M.; Liber, Howard L.

2005-01-01

Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

[Effect of increased protein content on nutritional and sensory quality of cookies].

PubMed

Pérez, Santiago Rafael; Osella, Carlos Alberto; Torre, Maria Adela de la; Sánchez, Hugo Diego

2008-12-01

The objective of this work was to study the effect of soy flour and whey protein concentrate (WPC) on cookies quality. An optimal recipe showing improved protein quality and content as well as acceptable sensory quality was defined taking into account the results obtained. Rotary moulded cookie formulation adaptable to lamination and cutting in pilot plant was used. Wheat flour from this formulation was partially replaced by whey protein concentrate and full fat soy flour. Second order models were employed to generate response surfaces for: total protein, lysine by 16 grams of total nitrogen, lysine by 100 grams of sample, loss of lysine during processing and sensory evaluation of cookies. We could obtain an effect on available lysine value when water content was increased in the formulation because a delay in the Maillard reaction. The optimal formulation contains 13% of full fat soy flour, 3% of whey protein concentrate and 23% of water. The results demonstrated that the protein content and the protein quality of the supplemented flours were increased when soy flour was added in the formulation of cookies. On other hand, protein content was increased but protein quality was decreased when WPC was used, because of available lysine loss.

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

PubMed Central

Maisnier-Patin, Sophie; Roth, John R.

2015-01-01

Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac+) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F′lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F′lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac+ triggers exponential cell growth leading to a stable Lac+ revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac+ colony. Cells with multiple copies of the F′lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac+ revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns–Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions. PMID:26134316

Variations in seed protein content of cotton (Gossypium hirsutum L.) mutant lines by in vivo and in vitro mutagenesis.

PubMed

Muthusamy, Annamalai; Jayabalan, Narayanasamy

2013-01-01

The present work describes the influence of gamma irradiation (GR), ethyl methane sulphonate (EMS) and sodium azide (SA) treatment on yield and protein content of selected mutant lines of cotton. Seeds of MCU 5 and MCU 11 were exposed to gamma rays (GR), ethyl methane sulphonate (EMS) and sodium azide (SA). Lower dose of gamma irradiation (100-500 Gy), 10-50 mM EMS and SA at lower concentration effectively influences in improving the yield and protein content. Significant increase in yield (258.9 g plant(-1)) and protein content (18.63 mg g(-1) d. wt.) as compared to parental lines was noted in M2 generations. During the subsequent field trials, number of mutant lines varied morphologically in terms of yield as well as biochemical characters such as protein. The selected mutant lines were bred true to their characters in M3 and M4 generations. The significant increase in protein content and profiles of the mutant lines with range of 10.21-18.63 mg g(-1). The SDS-PAGE analysis of mutant lines revealed 9 distinct bands of different intensities with range of 26-81 kDa. The difference in intensity of bands was more (41, 50 and 58 kDa) in the mutant lines obtained from in vitro mutation than in vivo mutation. Significance of such stimulation in protein content correlated with yielding ability of the mutant lines of cotton in terms of seed weight per plant. The results confirm that in cotton it is possible to enhance the both yield and biochemical characters by in vivo and in vitro mutagenic treatments.

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

PubMed Central

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

2016-01-01

Mutations in SMAD4 predispose to the development of gastrointestinal cancer, which is the third leading cause of cancer-related deaths. To identify genes driving gastric cancer (GC) development, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in the stomach of Smad4+/− mutant mice. This screen identified 59 candidate GC trunk drivers and a much larger number of candidate GC progression genes. Strikingly, 22 SB-identified trunk drivers are known or candidate cancer genes, whereas four SB-identified trunk drivers, including PTEN, SMAD4, RNF43, and NF1, are known human GC trunk drivers. Similar to human GC, pathway analyses identified WNT, TGF-β, and PI3K-PTEN signaling, ubiquitin-mediated proteolysis, adherens junctions, and RNA degradation in addition to genes involved in chromatin modification and organization as highly deregulated pathways in GC. Comparative oncogenomic filtering of the complete list of SB-identified genes showed that they are highly enriched for genes mutated in human GC and identified many candidate human GC genes. Finally, by comparing our complete list of SB-identified genes against the list of mutated genes identified in five large-scale human GC sequencing studies, we identified LDL receptor-related protein 1B (LRP1B) as a previously unidentified human candidate GC tumor suppressor gene. In LRP1B, 129 mutations were found in 462 human GC samples sequenced, and LRP1B is one of the top 10 most deleted genes identified in a panel of 3,312 human cancers. SB mutagenesis has, thus, helped to catalog the cooperative molecular mechanisms driving SMAD4-induced GC growth and discover genes with potential clinical importance in human GC. PMID:27006499

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

PubMed

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

2016-04-05

Mutations in SMAD4 predispose to the development of gastrointestinal cancer, which is the third leading cause of cancer-related deaths. To identify genes driving gastric cancer (GC) development, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in the stomach of Smad4(+/-) mutant mice. This screen identified 59 candidate GC trunk drivers and a much larger number of candidate GC progression genes. Strikingly, 22 SB-identified trunk drivers are known or candidate cancer genes, whereas four SB-identified trunk drivers, including PTEN, SMAD4, RNF43, and NF1, are known human GC trunk drivers. Similar to human GC, pathway analyses identified WNT, TGF-β, and PI3K-PTEN signaling, ubiquitin-mediated proteolysis, adherens junctions, and RNA degradation in addition to genes involved in chromatin modification and organization as highly deregulated pathways in GC. Comparative oncogenomic filtering of the complete list of SB-identified genes showed that they are highly enriched for genes mutated in human GC and identified many candidate human GC genes. Finally, by comparing our complete list of SB-identified genes against the list of mutated genes identified in five large-scale human GC sequencing studies, we identified LDL receptor-related protein 1B (LRP1B) as a previously unidentified human candidate GC tumor suppressor gene. In LRP1B, 129 mutations were found in 462 human GC samples sequenced, and LRP1B is one of the top 10 most deleted genes identified in a panel of 3,312 human cancers. SB mutagenesis has, thus, helped to catalog the cooperative molecular mechanisms driving SMAD4-induced GC growth and discover genes with potential clinical importance in human GC.

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

PubMed

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

2016-06-01

Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). Suicide substrates, such as ethyl or methyl bromobutyrate or alcohol dehydrogenase inhibitors like allyl alcohol, were tested and, finally, 36 mutants were isolated. The fermentation profiles of these NTG mutant strains were characterized, and the best performing mutants were used for consecutive rounds of genome shuffling. Screening of strains with further enhancement in isopropanol tolerance at each recursive shuffling step was then used to spot additionally improved strains. Three highly tolerant strains were finally isolated and able to withstand up to 50 g/L isopropanol on plates. Even if increased tolerance to the desired end product was not always accompanied by higher production capabilities, some shuffled strains showed increased solvent titers compared to the parental strains and the original C. beijerinckii DSM 6423. This study confirms the efficiency of genome shuffling to generate improved strains toward a desired phenotype such as alcohol tolerance. This tool also offers the possibility of obtaining improved strains of Clostridium species for which targeted genetic engineering approaches have not been described yet.

Ultrafast Hydration Dynamics and Coupled Water-Protein Fluctuations in Apomyoglobin

NASA Astrophysics Data System (ADS)

Yang, Yi; Zhang, Luyuan; Wang, Lijuan; Zhong, Dongping

2009-06-01

Protein hydration dynamics are of fundamental importance to its structure and function. Here, we characterize the global solvation dynamics and anisotropy dynamics around the apomyoglobin surface in different conformational states (native and molten globule) by measuring the Stokes shift and anisotropy decay of tryptophan with femtosecond-resolved fluorescence upconversion. With site-directed mutagenesis, we designed sixteen mutants with one tryptophan in each, and placed the probe at a desirable position ranging from buried in the protein core to fully solvent-exposed on the protein surface. In all protein sites studied, two distinct solvation relaxations (1-8 ps and 20-200 ps) were observed, reflecting the initial collective water relaxation and subsequent hydrogen-bond network restructuring, respectively, and both are strongly correlated with protein's local structures and chemical properties. The hydration dynamics of the mutants in molten globule state are faster than those observed in native state, indicating that the protein becomes more flexible and less structured when its conformation is converted from fully-folded native state to partially-folded molten globule state. Complementary, fluorescence anisotropy dynamics of all mutants in native state show an increasing trend of wobbling times (40-260 ps) when the location of the probe is changed from a loop, to a lateral helix, and then, to the compact protein core. Such an increase in wobbling times is related to the local protein structural rigidity, which relates the interaction of water with side chains. The ultrafast hydration dynamics and related side-chain motion around the protein surface unravel the coupled water-protein fluctuations on the picosecond time scales and indicate that the local protein motions are slaved by hydrating water fluctuations.

Cationic Antimicrobial Peptides Promote Microbial Mutagenesis and Pathoadaptation in Chronic Infections

PubMed Central

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

2014-01-01

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

Photoswitchable red fluorescent protein with a large Stokes shift.

PubMed

Piatkevich, Kiryl D; English, Brian P; Malashkevich, Vladimir N; Xiao, Hui; Almo, Steven C; Singer, Robert H; Verkhusha, Vladislav V

2014-10-23

A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622 nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621 nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.

Modification of mutagenesis initiated by ultraviolet light through posttreatment of bacteria with basic dyes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Witkin, E M

1961-12-01

Post treatment with basic dyes, in concentrations that retard cell division, was found to influence the tnduction of mutations to prototrophy by UV light in a tyrosine-requirtng strain of E.Coli. Pyronin, which is unique among the dyes in tts selective affinity for RNA, was found to duplicate the effects of chloramphenicol or amino acfd deprtvatfon in causfng the rapid and irreversible loss of potential prototrophs (mutation frequency decline, or MFD). Acriflavtne, methyl green. crystal violet, methylene blue, and toluidine blue, all of which are known to combine with DNA, delay or retard the occurrence of MFD under conditions of aminomore » acid deprivation. When acriflavine is removed from its combination with cellular components by the addition of an excess of sodium deoxyrtbonucleate, MFD begins promptly. The same basic dyes that delay MFD were also found to interfere with the fixation of mutations (MF) in an amino acid- enriched medium, and to cause marked enhancement of the mutagenic potency of low doses of UV light. While showing no independent mutagenic activity for unirradiated bacteria, all the dyes except pyronin increased the yield of induced mutations signtficantly when added to the enriched medium upon which trradiated bacteria were incubated.These results were interpreted as evidence that UV light initiates mutagenesis by producing unstable changas directly in genic DNA. MFD is interpreted as a repair process, blocked by the machinery of RNA and protein synthesis and by the presence of certain basic dyes.« less

Characterization of highly efficient heavy-ion mutagenesis in Arabidopsis thaliana.

PubMed

Kazama, Yusuke; Hirano, Tomonari; Saito, Hiroyuki; Liu, Yang; Ohbu, Sumie; Hayashi, Yoriko; Abe, Tomoko

2011-11-15

Heavy-ion mutagenesis is recognised as a powerful technology to generate new mutants, especially in higher plants. Heavy-ion beams show high linear energy transfer (LET) and thus more effectively induce DNA double-strand breaks than other mutagenic techniques. Previously, we determined the most effective heavy-ion LET (LETmax: 30.0 keV μm(-1)) for Arabidopsis mutagenesis by analysing the effect of LET on mutation induction. However, the molecular structure of mutated DNA induced by heavy ions with LETmax remains unclear. Knowledge of the structure of mutated DNA will contribute to the effective exploitation of heavy-ion beam mutagenesis. Dry Arabidopsis thaliana seeds were irradiated with carbon (C) ions with LETmax at a dose of 400 Gy and with LET of 22.5 keV μm(-1) at doses of 250 Gy or 450 Gy. The effects on mutation frequency and alteration of DNA structure were compared. To characterise the structure of mutated DNA, we screened the well-characterised mutants elongated hypocotyls (hy) and glabrous (gl) and identified mutated DNA among the resulting mutants by high-resolution melting curve, PCR and sequencing analyses. The mutation frequency induced by C ions with LETmax was two-fold higher than that with 22.5 keV μm(-1) and similar to the mutation frequency previously induced by ethyl methane sulfonate. We identified the structure of 22 mutated DNAs. Over 80% of the mutations caused by C ions with both LETs were base substitutions or deletions/insertions of less than 100 bp. The other mutations involved large rearrangements. The C ions with LETmax showed high mutation efficiency and predominantly induced base substitutions or small deletions/insertions, most of which were null mutations. These small alterations can be determined by single-nucleotide polymorphism (SNP) detection systems. Therefore, C ions with LETmax might be useful as a highly efficient reverse genetic system in conjunction with SNP detection systems, and will be beneficial for forward

EMSA Analysis of DNA Binding By Rgg Proteins.

PubMed

LaSarre, Breah; Federle, Michael J

2013-08-20

In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function ( e.g. interruption of DNA-binding in some cases).

Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes.

PubMed

Hanski, E; Caparon, M

1992-07-01

Binding to fibronectin has been suggested to play an important role in adherence of the group A streptococcus Streptococcus pyrogenes to host epithelial cells; however, the identity of the streptococcal fibronectin receptor has been elusive. Here we demonstrate that the fibronectin-binding property of S. pyogenes is mediated by protein F, a bacterial surface protein that binds fibronectin at high affinity. The gene encoding protein F (prtF) produced a functional fibronectin-binding protein in Escherichia coli. Insertional mutagenesis of the cloned gene generated a mutation that resulted in the loss of fibronectin-binding activity. When this mutation was introduced into the S. pyrogenes chromosome by homologous recombination with the wild-type allele, the resulting strains no longer produced protein F and lost their ability to bind fibronectin. The mutation could be complemented by prtF introduced on a plasmid. Mutants lacking protein F had a much lower capacity to adhere to respiratory epithelial cells. These results demonstrate that protein F is an important adhesin of S. pyogenes.

One-step combined focused epPCR and saturation mutagenesis for thermostability evolution of a new cold-active xylanase.

PubMed

Acevedo, Juan Pablo; Reetz, Manfred T; Asenjo, Juan A; Parra, Loreto P

2017-05-01

Enzymes active at low temperature are of great interest for industrial bioprocesses due to their high efficiency at a low energy cost. One of the particularities of naturally evolved cold-active enzymes is their increased enzymatic activity at low temperature, however the low thermostability presented in this type of enzymes is still a major drawback for their application in biocatalysis. Directed evolution of cold-adapted enzymes to a more thermostable version, appears as an attractive strategy to fulfill the stability and activity requirements for the industry. This paper describes the recombinant expression and characterization of a new and highly active cold-adapted xylanase from the GH-family 10 (Xyl-L), and the use of a novel one step combined directed evolution technique that comprises saturation mutagenesis and focused epPCR as a feasible semi-rational strategy to improve the thermostability. The Xyl-L enzyme was cloned from a marine-Antarctic bacterium, Psychrobacter sp. strain 2-17, recombinantly expressed in E. coli strain BL21(DE3) and characterized enzymatically. Molecular dynamic simulations using a homology model of the catalytic domain of Xyl-L were performed to detect flexible regions and residues, which are considered to be the possible structural elements that define the thermolability of this enzyme. Mutagenic libraries were designed in order to stabilize the protein introducing mutations in some of the flexible regions and residues identified. Twelve positive mutant clones were found to improve the T 50 15 value of the enzyme, in some cases without affecting the activity at 25°C. The best mutant showed a 4.3°C increase in its T 50 15 . The efficiency of the directed evolution approach can also be expected to work in the protein engineering of stereoselectivity. Copyright © 2017 Elsevier Inc. All rights reserved.

Increasing dietary protein requirements in elderly people for optimal muscle and bone health.

PubMed

Gaffney-Stomberg, Erin; Insogna, Karl L; Rodriguez, Nancy R; Kerstetter, Jane E

2009-06-01

Osteoporosis and sarcopenia are degenerative diseases frequently associated with aging. The loss of bone and muscle results in significant morbidity, so preventing or attenuating osteoporosis and sarcopenia is an important public health goal. Dietary protein is crucial for development of bone and muscle, and recent evidence suggests that increasing dietary protein above the current Recommended Dietary Allowance (RDA) may help maintain bone and muscle mass in older individuals. Several epidemiological and clinical studies point to a salutary effect of protein intakes above the current RDA (0.8 g/kg per day) for adults aged 19 and older. There is evidence that the anabolic response of muscle to dietary protein is attenuated in elderly people, and as a result, the amount of protein needed to achieve anabolism is greater. Dietary protein also increases circulating insulin-like growth factor, which has anabolic effects on muscle and bone. Furthermore, increasing dietary protein increases calcium absorption, which could be anabolic for bone. Available evidence supports a beneficial effect of short-term protein intakes up to 1.6 to 1.8 g/kg per day, although long-term studies are needed to show safety and efficacy. Future studies should employ functional measures indicative of protein adequacy, as well as measures of muscle protein synthesis and maintenance of muscle and bone tissue, to determine the optimal level of dietary protein. Given the available data, increasing the RDA for older individuals to 1.0 to 1.2 g/kg per day would maintain normal calcium metabolism and nitrogen balance without affecting renal function and may represent a compromise while longer-term protein supplement trials are pending.

Acute supplementation of amino acids increases net protein accretion in IUGR fetal sheep

PubMed Central

Rozance, Paul J.; Thorn, Stephanie R.; Friedman, Jacob E.; Hay, William W.

2012-01-01

Placental insufficiency decreases fetal amino acid uptake from the placenta, plasma insulin concentrations, and protein accretion, thus compromising normal fetal growth trajectory. We tested whether acute supplementation of amino acids or insulin into the fetus with intrauterine growth restriction (IUGR) would increase net fetal protein accretion rates. Late-gestation IUGR and control (CON) fetal sheep received acute, 3-h infusions of amino acids (with euinsulinemia), insulin (with euglycemia and euaminoacidemia), or saline. Fetal leucine metabolism was measured under steady-state conditions followed by a fetal muscle biopsy to quantify insulin signaling. In CON, increasing amino acid delivery rates to the fetus by 100% increased leucine oxidation rates by 100%. In IUGR, amino acid infusion completely suppressed fetal protein breakdown rates but increased leucine oxidation rate by only 25%, resulting in increased protein accretion rates by 150%. Acute insulin infusion, however, had very little effect on amino acid delivery rates, fetal leucine disposal rates, or fetal protein accretion rates in CON or IUGR fetuses despite robust signaling of the fetal skeletal muscle insulin-signaling cascade. These results indicate that, when amino acids are given directly into the fetal circulation independently of changes in insulin concentrations, IUGR fetal sheep have suppressed protein breakdown rates, thus increasing net fetal protein accretion. PMID:22649066

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

PubMed Central

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

2015-01-01

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

The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.

PubMed

Adu-Gyamfi, Emmanuel; Soni, Smita P; Xue, Yi; Digman, Michelle A; Gratton, Enrico; Stahelin, Robert V

2013-02-22

Ebola, a fatal virus in humans and non-human primates, has no Food and Drug Administration-approved vaccines or therapeutics. The virus from the Filoviridae family causes hemorrhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes, the most abundantly expressed of which is viral protein 40 (VP40), the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the plasma membrane; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of biophysical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal domain. Mutagenesis of this hydrophobic region consisting of Leu(213), Ile(293), Leu(295), and Val(298) demonstrated that membrane penetration is critical to plasma membrane localization, VP40 oligomerization, and viral particle egress. Taken together, VP40 membrane penetration is an important step in the plasma membrane localization of the matrix protein where oligomerization and budding are defective in the absence of key hydrophobic interactions with the membrane.

Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains.

PubMed

Shi, Junwei; Wang, Eric; Milazzo, Joseph P; Wang, Zihua; Kinney, Justin B; Vakoc, Christopher R

2015-06-01

CRISPR-Cas9 genome editing technology holds great promise for discovering therapeutic targets in cancer and other diseases. Current screening strategies target CRISPR-Cas9-induced mutations to the 5' exons of candidate genes, but this approach often produces in-frame variants that retain functionality, which can obscure even strong genetic dependencies. Here we overcome this limitation by targeting CRISPR-Cas9 mutagenesis to exons encoding functional protein domains. This generates a higher proportion of null mutations and substantially increases the potency of negative selection. We also show that the magnitude of negative selection can be used to infer the functional importance of individual protein domains of interest. A screen of 192 chromatin regulatory domains in murine acute myeloid leukemia cells identifies six known drug targets and 19 additional dependencies. A broader application of this approach may allow comprehensive identification of protein domains that sustain cancer cells and are suitable for drug targeting.

Lysine Methylation of Nuclear Co-repressor Receptor Interacting Protein 140

PubMed Central

Huq, MD Mostaqul; Ha, Sung Gil; Barcelona, Helene; Wei, Li-Na

2009-01-01

Receptor interacting protein 140 (RIP140) undergoes extensive posttranslational modifications (PTMs), including phosphorylation, acetylation, arginine methylation, and pyridoxylation. PTMs affect its sub-cellular distribution, protein-protein interaction, and biological activity in adipocyte differentiation. Arginine methylation on Arg240, Arg650, and Arg948 suppresses the repressive activity of RIP140. Here we find that endogenous RIP140 in differentiated 3T3-L1 cells is also modified by lysine methylation. Three lysine residues, Lys591, Lys653, and Lys757 are mapped as potential methylation sites by mass spectrometry. Site-directed mutagenesis study shows that lysine methylation enhances its gene repressive activity. Mutation of lysine methylation sites enhances arginine methylation, while mutation on arginine methylation sites has little effect on its lysine methylation, suggesting a relationship between lysine methylation and arginine methylation. Kinetic analysis of PTMs of endogenous RIP140 in differentiated 3T3-L1 cells demonstrates sequential modifications on RIP140, initiated from constitutive lysine methylation, followed by increased arginine methylation later in differentiation. This study reveals a potential hierarchy of modifications, at least for lysine and arginine methylation, which bi-directionally regulate the functionality of a non-histone protein. PMID:19216533

Protein consensus-based surface engineering (ProCoS): a computer-assisted method for directed protein evolution.

PubMed

Shivange, Amol V; Hoeffken, Hans Wolfgang; Haefner, Stefan; Schwaneberg, Ulrich

2016-12-01

Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: ( i ) identification of conserved and highly variable regions; ( ii ) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; ( iii ) in vitro DNA recombination of synthetic genes; and ( iv ) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.

The SOS response increases bacterial fitness, but not evolvability, under a sublethal dose of antibiotic.

PubMed

Torres-Barceló, Clara; Kojadinovic, Mila; Moxon, Richard; MacLean, R Craig

2015-10-07

Exposure to antibiotics induces the expression of mutagenic bacterial stress-response pathways, but the evolutionary benefits of these responses remain unclear. One possibility is that stress-response pathways provide a short-term advantage by protecting bacteria against the toxic effects of antibiotics. Second, it is possible that stress-induced mutagenesis provides a long-term advantage by accelerating the evolution of resistance. Here, we directly measure the contribution of the Pseudomonas aeruginosa SOS pathway to bacterial fitness and evolvability in the presence of sublethal doses of ciprofloxacin. Using short-term competition experiments, we demonstrate that the SOS pathway increases competitive fitness in the presence of ciprofloxacin. Continued exposure to ciprofloxacin results in the rapid evolution of increased fitness and antibiotic resistance, but we find no evidence that SOS-induced mutagenesis accelerates the rate of adaptation to ciprofloxacin during a 200 generation selection experiment. Intriguingly, we find that the expression of the SOS pathway decreases during adaptation to ciprofloxacin, and this helps to explain why this pathway does not increase long-term evolvability. Furthermore, we argue that the SOS pathway fails to accelerate adaptation to ciprofloxacin because the modest increase in the mutation rate associated with SOS mutagenesis is offset by a decrease in the effective strength of selection for increased resistance at a population level. Our findings suggest that the primary evolutionary benefit of the SOS response is to increase bacterial competitive ability, and that stress-induced mutagenesis is an unwanted side effect, and not a selected attribute, of this pathway. © 2015 The Authors.

The SOS response increases bacterial fitness, but not evolvability, under a sublethal dose of antibiotic

PubMed Central

Torres-Barceló, Clara; Kojadinovic, Mila; Moxon, Richard; MacLean, R. Craig

2015-01-01

Exposure to antibiotics induces the expression of mutagenic bacterial stress–response pathways, but the evolutionary benefits of these responses remain unclear. One possibility is that stress–response pathways provide a short-term advantage by protecting bacteria against the toxic effects of antibiotics. Second, it is possible that stress-induced mutagenesis provides a long-term advantage by accelerating the evolution of resistance. Here, we directly measure the contribution of the Pseudomonas aeruginosa SOS pathway to bacterial fitness and evolvability in the presence of sublethal doses of ciprofloxacin. Using short-term competition experiments, we demonstrate that the SOS pathway increases competitive fitness in the presence of ciprofloxacin. Continued exposure to ciprofloxacin results in the rapid evolution of increased fitness and antibiotic resistance, but we find no evidence that SOS-induced mutagenesis accelerates the rate of adaptation to ciprofloxacin during a 200 generation selection experiment. Intriguingly, we find that the expression of the SOS pathway decreases during adaptation to ciprofloxacin, and this helps to explain why this pathway does not increase long-term evolvability. Furthermore, we argue that the SOS pathway fails to accelerate adaptation to ciprofloxacin because the modest increase in the mutation rate associated with SOS mutagenesis is offset by a decrease in the effective strength of selection for increased resistance at a population level. Our findings suggest that the primary evolutionary benefit of the SOS response is to increase bacterial competitive ability, and that stress-induced mutagenesis is an unwanted side effect, and not a selected attribute, of this pathway. PMID:26446807

Efficient Mutagenesis Independent of Ligation (EMILI).

PubMed

Füzik, Tibor; Ulbrich, Pavel; Ruml, Tomáš

2014-11-01

Site-directed mutagenesis is one of the most widely used techniques in life sciences. Here we describe an improved and simplified method for introducing mutations at desired sites. It consists of an inverse PCR using a plasmid template and two partially complementary primers. The synthesis step is followed by annealing of the PCR product's sticky ends, which are generated by exonuclease digestion. This method is fast, extremely efficient and cost-effective. It can be used to introduce large insertions and deletions, but also for multiple point mutations in a single step. To show the principle and to prove the efficiency of the method, we present a series of basic mutations (insertions, deletions, point mutations) on pUC19 plasmid DNA. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of amino acid residues responsible for differences in substrate specificity and inhibitor sensitivity between two human liver dihydrodiol dehydrogenase isoenzymes by site-directed mutagenesis.

PubMed Central

Matsuura, K; Deyashiki, Y; Sato, K; Ishida, N; Miwa, G; Hara, A

1997-01-01

Human liver dihydrodiol dehydrogenase isoenzymes (DD1 and DD2), in which only seven amino acid residues are substituted, differ remarkably in specificity for steroidal substrates and inhibitor sensitivity: DD1 shows 20alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas DD2 oxidizes 3alpha-hydroxysteroids and is highly inhibited by bile acids. In the present study we performed site-directed mutagenesis of the seven residues (Thr-38, Arg-47, Leu-54, Cys-87, Val-151, Arg-170 and Gln-172) of DD1 to the corresponding residues (Val, His, Val, Ser, Met, His and Leu respectively) of DD2. Of the seven mutations, only the replacement of Leu-54 with Val produced an enzyme that had almost the same properties as DD2. No significant changes were observed in the other mutant enzymes. An additional site-directed mutagenesis of Tyr-55 of DD1 to Phe yielded an inactive protein, suggesting the catalytically important role of this residue. Thus a residue at a position before the catalytic Tyr residue might play a key role in determining the orientation of the substrates and inhibitors. PMID:9173902

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody.

PubMed

Monnet, Céline; Jorieux, Sylvie; Souyris, Nathalie; Zaki, Ouafa; Jacquet, Alexandra; Fournier, Nathalie; Crozet, Fabien; de Romeuf, Christophe; Bouayadi, Khalil; Urbain, Rémi; Behrens, Christian K; Mondon, Philippe; Fontayne, Alexandre

2014-01-01

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGen™) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling(®) platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.

Fluorescence Modulation of Green Fluorescent Protein Using Fluorinated Unnatural Amino Acids.

PubMed

Villa, Jordan K; Tran, Hong-Anh; Vipani, Megha; Gianturco, Stephanie; Bhasin, Konark; Russell, Brent L; Harbron, Elizabeth J; Young, Douglas D

2017-07-16

The ability to modulate protein function through minimal perturbations to amino acid structure represents an ideal mechanism to engineer optimized proteins. Due to the novel spectroscopic properties of green fluorescent protein, it has found widespread application as a reporter protein throughout the fields of biology and chemistry. Using site-specific amino acid mutagenesis, we have incorporated various fluorotyrosine residues directly into the fluorophore of the protein, altering the fluorescence and shifting the pKa of the phenolic proton associated with the fluorophore. Relative to wild type GFP, the fluorescence spectrum of the protein is altered with each additional fluorine atom, and the mutant GFPs have the potential to be employed as pH sensors due to the altered electronic properties of the fluorine atoms.

Effect of increasing dietary metabolizable protein on nitrogen efficiency in Holstein dairy cows

PubMed Central

Imran, Muhammad; Pasha, Talat Naseer; Shahid, Muhammad Qamer; Babar, Imran; Naveed ul Haque, Muhammad

2017-01-01

Objective The objective of the study was to determine the effects of increasing levels of metabolizable protein (MP) on lactation performance and nitrogen (N) efficiencies in lactating dairy cows. Methods Nine multiparous cows in mid lactation [113±25 days in milk] received three treatments in a 3×3 Latin square design with a period length of 21 days. The treatments were three diets, designed to provide similar energy and increasing supply of MP (g/d) (2,371 [low], 2,561 [medium], and 2,711 [high] with corresponding crude protein levels [%]) 15.2, 18.4, and 20.9, respectively. Results Increasing MP supplies did not modify dry matter intake, however, it increased milk protein, fat, and lactose yield linearly. Similarly, fat corrected milk increased linearly (9.3%) due to an increase in both milk yield (5.2%) and milk fat content (7.8%). No effects were observed on milk protein and lactose contents across the treatments. Milk nitrogen efficiency (MNE) decreased from 0.26 to 0.20; whereas, the metabolic efficiency of MP decreased from 0.70 to 0.60 in low to high MP supplies, respectively. The concentration of blood urea nitrogen (BUN) increased linearly in response to increasing MP supplies. Conclusion Increasing MP supplies resulted in increased milk protein yield; however, a higher BUN and low MNE indicated an efficient utilization of dietary protein at low MP supplies. PMID:28002937

Insertion and deletion mutagenesis of the human cytomegalovirus genome

DOE Office of Scientific and Technical Information (OSTI.GOV)

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, withmore » 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.« less

Genome-Wide Transposon Mutagenesis in Pathogenic Leptospira Species▿ ‡

PubMed Central

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

2009-01-01

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

The Structure of Urease Activation Complexes Examined by Flexibility Analysis, Mutagenesis, and Small-Angle X-Ray Scattering

PubMed Central

Quiroz-Valenzuela, Soledad; Sukuru, Sai Chetan K.; Hausinger, Robert P.; Kuhn, Leslie A.; Heller, William T.

2008-01-01

Conformational changes of Klebsiella aerogenes urease apoprotein (UreABC)3 induced upon binding of the UreD and UreF accessory proteins were examined by a combination of flexibility analysis, mutagenesis, and small-angle x-ray scattering (SAXS). ProFlex analysis of urease provided evidence that the major domain of UreB can move in a hinge-like motion to account for prior chemical cross-linking results. Rigidification of the UreB hinge region, accomplished through a G11P mutation, reduced the extent of urease activation, in part by decreasing the nickel content of the mutant enzyme, and by sequestering a portion of the urease apoprotein in a novel activation complex that includes all of the accessory proteins. SAXS analyses of urease, (UreABC-UreD)3, and (UreABC-UreDF)3 confirm that UreD and UreF bind near UreB at the periphery of the (UreAC)3 structure. This study supports an activation model in which a domain-shifted UreB conformation in (UreABC-UreDF)3 allows CO2 and nickel ions to gain access to the nascent active site. PMID:18823937

A comparison of bactericidal/permeability-increasing protein variant versus recombinant endotoxin-neutralizing protein for the treatment of Escherichia coli sepsis in rats .

PubMed

Stack, A M; Saladino, R A; Siber, G R; Thompson, C; Marra, M N; Novitsky, T J; Fleisher, G R

1997-01-01

To compare a recombinant bactericidal/permeability-increasing protein variant and a recombinant endotoxin-neutralizing protein. Randomized, blinded, controlled study, using a rat model of sepsis. Animal research facility. Male Wistar rats. An inoculum of 1.5 x 10(7) to 1.8 x 10(8) Escherichia coli O18ac K1, implanted in the peritoneum, produced bacteremia in 95% of animals after 1 hr. One hour after E. coli challenge, animals received recombinant bactericidal/permeability-increasing protein variant, recombinant endotoxin-neutralizing protein, or saline intravenously, followed by ceftriaxone and gentamicin intramuscularly. Twenty-four (85.7%) of 28 animals receiving recombinant endotoxin-neutralizing protein (p < .001 vs. control) survived 7 days compared with nine (33.3%) of 27 recombinant bactericidal/permeability-increasing protein variant-treated (p < .001 vs. control) and two (6.5%) of 31 control animals. Both recombinant endotoxin-neutralizing protein and recombinant bactericidal/permeability-increasing protein variant improved survival. Recombinant endotoxin-neutralizing protein was superior to recombinant bactericidal/permeability-increasing protein variant in its protective effect at the doses tested. Our results suggest that both proteins may be useful in the treatment of human Gram-negative sepsis.

Method For Determining And Modifying Protein/Peptide Solubilty

DOEpatents

Waldo, Geoffrey S.

2005-03-15

A solubility reporter for measuring a protein's solubility in vivo or in vitro is described. The reporter, which can be used in a single living cell, gives a specific signal suitable for determining whether the cell bears a soluble version of the protein of interest. A pool of random mutants of an arbitrary protein, generated using error-prone in vitro recombination, may also be screened for more soluble versions using the reporter, and these versions may be recombined to yield variants having further-enhanced solubility. The method of the present invention includes "irrational" (random mutagenesis) methods, which do not require a priori knowledge of the three-dimensional structure of the protein of interest. Multiple sequences of mutation/genetic recombination and selection for improved solubility are demonstrated to yield versions of the protein which display enhanced solubility.

Amplification of the groESL operon in Pseudomonas putida increases siderophore gene promoter activity.

PubMed

Venturi, V; Wolfs, K; Leong, J; Weisbeek, P J

1994-10-17

Pseudobactin 358 is the yellow-green fluorescent siderophore [microbial iron(III) transport agent] produced by Pseudomonas putida WCS358 under iron-limiting conditions. The genes encoding pseudobactin 358 biosynthesis are iron-regulated at the level of transcription. In this study, the molecular characterization is reported of a cosmid clone of WCS358 DNA that can stimulate, in an iron-dependent manner, the activity of a WCS358 siderophore gene promoter in the heterologous Pseudomonas strain A225. The functional region in the clone was identified by subcloning, transposon mutagenesis and DNA sequencing as the groESL operon of strain WCS358. This increase in promoter activity was not observed when the groESL genes of strain WCS358 were integrated via a transposon vector into the genome of Pseudomonas A225, indicating that multiple copies of the operon are necessary for the increase in siderophore gene promoter activity. Amplification of the Escherichia coli and WCS358 groESL genes also increased iron-regulated promoter activity in the parent strain WCS358. The groESL operon codes for the chaperone proteins GroES and GroEL, which are responsible for mediating the folding and assembly of many proteins.

Increased Temperature and Protein Oxidation Signal HSP72 mRNA and Protein Accumulation in the In Vivo Exercised Rat Heart

PubMed Central

Staib, Jessica L.; Tümer, Nihal; Powers, Scott K.

2010-01-01

Myocardial heat shock protein 72 (HSP72) expression, mediated by its transcription factor heat shock factor 1 (HSF1), increases following exercise. However, the up-stream stimuli governing exercise-induced HSF1 activation and subsequent HSP72 gene expression in the whole animal remain unclear. Exercise-induced increases in body temperature may promote myocardial radical production leading to protein oxidation. Conceivably, myocardial protein oxidation during exercise may serve as an important signal promoting nuclear HSF1 migration and activation of HSP72 expression. Therefore, these experiments tested the hypothesis that preventing exercise-induced increases in body temperature attenuates cardiac protein oxidation, diminishes HSF1 activation and decreases HSP72 expression in vivo. To test this hypothesis, in vivo exercise-induced body temperature was manipulated by exercising male rats in either cold (4°C) or warm (22°C) ambient conditions. Warm exercise increased both body temperature (+ 3°C) and myocardial protein oxidation whereas these changes were attenuated by cold exercise. Interestingly, exercise in both conditions did not significantly increase myocardial nuclear localized phosphorylated HSF1. Nonetheless, warm exercise elevated left-ventricular HSP72 mRNA by 9-fold and increased myocardial HSP72 protein levels by 3-fold compared to cold-exercised animals. Collectively, these data indicate that elevated body temperature and myocardial protein oxidation promoted exercise-induced cardiac HSP72 mRNA expression and protein accumulation following in vivo exercise. However, these results suggest that exercise-induced myocardial HSP72 protein accumulation is not a result of nuclear-localized, phosphorylated HSF1 indicating that other transcriptional or posttranscriptional regulatory mechanisms are involved in exercise-induced HSP72 expression. PMID:18931043

Prediction of protein interaction hot spots using rough set-based multiple criteria linear programming.

PubMed

Chen, Ruoying; Zhang, Zhiwang; Wu, Di; Zhang, Peng; Zhang, Xinyang; Wang, Yong; Shi, Yong

2011-01-21

Protein-protein interactions are fundamentally important in many biological processes and it is in pressing need to understand the principles of protein-protein interactions. Mutagenesis studies have found that only a small fraction of surface residues, known as hot spots, are responsible for the physical binding in protein complexes. However, revealing hot spots by mutagenesis experiments are usually time consuming and expensive. In order to complement the experimental efforts, we propose a new computational approach in this paper to predict hot spots. Our method, Rough Set-based Multiple Criteria Linear Programming (RS-MCLP), integrates rough sets theory and multiple criteria linear programming to choose dominant features and computationally predict hot spots. Our approach is benchmarked by a dataset of 904 alanine-mutated residues and the results show that our RS-MCLP method performs better than other methods, e.g., MCLP, Decision Tree, Bayes Net, and the existing HotSprint database. In addition, we reveal several biological insights based on our analysis. We find that four features (the change of accessible surface area, percentage of the change of accessible surface area, size of a residue, and atomic contacts) are critical in predicting hot spots. Furthermore, we find that three residues (Tyr, Trp, and Phe) are abundant in hot spots through analyzing the distribution of amino acids. Copyright © 2010 Elsevier Ltd. All rights reserved.

Antenatal dietary education and supplementation to increase energy and protein intake.

PubMed

Ota, Erika; Hori, Hiroyuki; Mori, Rintaro; Tobe-Gai, Ruoyan; Farrar, Diane

2015-06-02

Gestational weight gain is positively associated with fetal growth, and observational studies of food supplementation in pregnancy have reported increases in gestational weight gain and fetal growth. To assess the effects of education during pregnancy to increase energy and protein intake, or of actual energy and protein supplementation, on energy and protein intake, and the effect on maternal and infant health outcomes. We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (31 January 2015), reference lists of retrieved studies and contacted researchers in the field. Randomised controlled trials of dietary education to increase energy and protein intake, or of actual energy and protein supplementation, during pregnancy. Two review authors independently assessed trials for inclusion and assessed risk of bias. Two review authors independently extracted data and checked for accuracy. Extracted data were supplemented by additional information from the trialists we contacted. We examined 149 reports corresponding to 65 trials. Of these trials, 17 were included, 46 were excluded, and two are ongoing. Overall, 17 trials involving 9030 women were included. For this update, we assessed methodological quality of the included trials using the standard Cochrane criteria (risk of bias) and the GRADE approach. The overall risk of bias was unclear. Nutritional education (five trials, 1090 women) Women given nutritional education had a lower relative risk of having a preterm birth (two trials, 449 women) (risk ratio (RR) 0.46, 95% CI 0.21 to 0.98, low-quality evidence), and low birthweight (one trial, 300 women) (RR 0.04, 95% CI 0.01 to 0.14). Head circumference at birth was increased in one trial (389 women) (mean difference (MD) 0.99 cm, 95% CI 0.43 to 1.55), while birthweight was significantly increased among undernourished women in two trials (320 women) (MD 489.76 g, 95% CI 427.93 to 551.59, low-quality evidence), but did not significantly increase for

Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Rongming; Liang, Liya; Garst, Andrew D.

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

Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering

DOE PAGES

Liu, Rongming; Liang, Liya; Garst, Andrew D.; ...

2018-03-29

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

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

PubMed Central

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

2016-01-01

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

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

USDA-ARS?s Scientific Manuscript database

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

A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp. michiganensis.

PubMed

Kirchner, O; Gartemann, K H; Zellermann, E M; Eichenlaub, R; Burger, A

2001-11-01

A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 x 10(6) transformants per microg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 x 10(3) transposon mutants per pg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.

EMSA Analysis of DNA Binding By Rgg Proteins

PubMed Central

LaSarre, Breah; Federle, Michael J.

2016-01-01

In bacteria, interaction of various proteins with DNA is essential for the regulation of specific target gene expression. Electrophoretic mobility shift assay (EMSA) is an in vitro approach allowing for the visualization of these protein-DNA interactions. Rgg proteins comprise a family of transcriptional regulators widespread among the Firmicutes. Some of these proteins function independently to regulate target gene expression, while others have now been demonstrated to function as effectors of cell-to-cell communication, having regulatory activities that are modulated via direct interaction with small signaling peptides. EMSA analysis can be used to assess DNA binding of either type of Rgg protein. EMSA analysis of Rgg protein activity has facilitated in vitro confirmation of regulatory targets, identification of precise DNA binding sites via DNA probe mutagenesis, and characterization of the mechanism by which some cognate signaling peptides modulate Rgg protein function (e.g. interruption of DNA-binding in some cases). PMID:27430004

Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum.

PubMed Central

Rella, M; Axelrood, P E; Weinhold, A R; Schroth, M N

1989-01-01

The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled transposon mutagenesis of a wild-type antibiotic-producing strain of E. carotovora subsp. betavasculorum. Twelve antibiotic-negative mutants were isolated, and one of these showed a reduction in antibiotic production in vitro. Many of these mutants also showed a reduction in their ability to macerate potato tissue. The mutants were classified into four genetic groups on the basis of their genetic and phenotypic characteristics, indicating that several genes are involved in antibiotic biosynthesis by E. carotovora subsp. betavasculorum. PMID:2543291

The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis.

PubMed

Welsh, Sarah J; Bellamy, William T; Briehl, Margaret M; Powis, Garth

2002-09-01

Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

Cloning, Functional Characterization and Site-Directed Mutagenesis of 4-Coumarate: Coenzyme A Ligase (4CL) Involved in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn

PubMed Central

Liu, Tingting; Yao, Ruolan; Zhao, Yucheng; Xu, Sheng; Huang, Chuanlong; Luo, Jun; Kong, Lingyi

2017-01-01

Coumarins are the main bioactive compounds in Peucedanum praeruptorum Dunn, a common Chinese herbal medicine. Nevertheless, the genes involved in the biosynthesis of core structure of coumarin in P. praeruptorum have not been identified yet. 4-Coumarate: CoA ligase (4CL) catalyzes the formation of hydroxycinnamates CoA esters, and plays an essential role at the divergence point from general phenylpropanoid metabolism to major branch pathway of coumarin. Here, three novel putative 4CL genes (Pp4CL1, Pp4CL7, and Pp4CL10) were isolated from P. praeruptorum. Biochemical characterization of the recombinant proteins revealed that Pp4CL1 utilized p-coumaric and ferulic acids as its two main substrates for coumarin biosynthesis in P. praeruptorum. Furthermore, Pp4CL1 also exhibited activity toward caffeic, cinnamic, isoferulic, and o-coumaric acids and represented a bona fide 4CL. Pp4CL7 and Pp4CL10 had no catalytic activity toward hydroxycinnamic acid compounds. But they had close phylogenetic relationship to true 4CLs and were defined as 4CL-like genes. Among all putative 4CLs, Pp4CL1 was the most highly expressed gene in roots, and its expression level was significantly up-regulated in mature roots compared with seedlings. Subcellular localization studies showed that Pp4CL1 and Pp4CL10 proteins were localized in the cytosol. In addition, site-directed mutagenesis of Pp4CL1 demonstrated that amino acids of Tyr-239, Ala-243, Met-306, Ala-309, Gly-334, Lys-441, Gln-446, and Lys-526 were essential for substrate binding or catalytic activities. The characterization and site-directed mutagenesis studies of Pp4CL1 lays a solid foundation for elucidating the biosynthetic mechanisms of coumarins in P. praeruptorum and provides further insights in understanding the structure–function relationships of this important family of proteins. PMID:28144249

Identification of Genes Involved in Biofilm Formation and Respiration via Mini-Himar Transposon Mutagenesis of Geobacter sulfurreducens▿ †

PubMed Central

Rollefson, Janet B.; Levar, Caleb E.; Bond, Daniel R.

2009-01-01

Electron transfer from cells to metals and electrodes by the Fe(III)-reducing anaerobe Geobacter sulfurreducens requires proper expression of redox proteins and attachment mechanisms to interface bacteria with surfaces and neighboring cells. We hypothesized that transposon mutagenesis would complement targeted knockout studies in Geobacter spp. and identify novel genes involved in this process. Escherichia coli mating strains and plasmids were used to develop a conjugation protocol and deliver mini-Himar transposons, creating a library of over 8,000 mutants that was anaerobically arrayed and screened for a range of phenotypes, including auxotrophy for amino acids, inability to reduce Fe(III) citrate, and attachment to surfaces. Following protocol validation, mutants with strong phenotypes were further characterized in a three-electrode system to simultaneously quantify attachment, biofilm development, and respiratory parameters, revealing mutants defective in Fe(III) reduction but unaffected in electron transfer to electrodes (such as an insertion in GSU1330, a putative metal export protein) or defective in electrode reduction but demonstrating wild-type biofilm formation (due to an insertion upstream of the NHL domain protein GSU2505). An insertion in a putative ATP-dependent transporter (GSU1501) eliminated electrode colonization but not Fe(III) citrate reduction. A more complex phenotype was demonstrated by a mutant containing an insertion in a transglutaminase domain protein (GSU3361), which suddenly ceased to respire when biofilms reached approximately 50% of the wild-type levels. As most insertions were not in cytochromes but rather in transporters, two-component signaling proteins, and proteins of unknown function, this collection illustrates how biofilm formation and electron transfer are separate but complementary phenotypes, controlled by multiple loci not commonly studied in Geobacter spp. PMID:19395486

Structural insights into the recovery of aldolase activity in N-acetylneuraminic acid lyase by replacement of the catalytically active lysine with γ-thialysine by using a chemical mutagenesis strategy.

PubMed

Timms, Nicole; Windle, Claire L; Polyakova, Anna; Ault, James R; Trinh, Chi H; Pearson, Arwen R; Nelson, Adam; Berry, Alan

2013-03-04

Chemical modification has been used to introduce the unnatural amino acid γ-thialysine in place of the catalytically important Lys165 in the enzyme N-acetylneuraminic acid lyase (NAL). The Staphylococcus aureus nanA gene, encoding NAL, was cloned and expressed in E. coli. The protein, purified in high yield, has all the properties expected of a class I NAL. The S. aureus NAL which contains no natural cysteine residues was subjected to site-directed mutagenesis to introduce a cysteine in place of Lys165 in the enzyme active site. Subsequently chemical mutagenesis completely converted the cysteine into γ-thialysine through dehydroalanine (Dha) as demonstrated by ESI-MS. Initial kinetic characterisation showed that the protein containing γ-thialysine regained 17 % of the wild-type activity. To understand the reason for this lower activity, we solved X-ray crystal structures of the wild-type S. aureus NAL, both in the absence of, and in complex with, pyruvate. We also report the structures of the K165C variant, and the K165-γ-thialysine enzyme in the presence, or absence, of pyruvate. These structures reveal that γ-thialysine in NAL is an excellent structural mimic of lysine. Measurement of the pH-activity profile of the thialysine modified enzyme revealed that its pH optimum is shifted from 7.4 to 6.8. At its optimum pH, the thialysine-containing enzyme showed almost 30 % of the activity of the wild-type enzyme at its pH optimum. The lowered activity and altered pH profile of the unnatural amino acid-containing enzyme can be rationalised by imbalances of the ionisation states of residues within the active site when the pK(a) of the residue at position 165 is perturbed by replacement with γ-thialysine. The results reveal the utility of chemical mutagenesis for the modification of enzyme active sites and the exquisite sensitivity of catalysis to the local structural and electrostatic environment in NAL. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Virus-based Photo-Responsive Nanowires Formed By Linking Site-Directed Mutagenesis and Chemical Reaction

NASA Astrophysics Data System (ADS)

Murugesan, Murali; Abbineni, Gopal; Nimmo, Susan L.; Cao, Binrui; Mao, Chuanbin

2013-05-01

Owing to the genetic flexibility and error-free bulk production, bio-nanostructures such as filamentous phage showed great potential in materials synthesis, however, their photo-responsive behaviour is neither explored nor unveiled. Here we show M13 phage genetically engineered with tyrosine residues precisely fused to the major coat protein is converted into a photo-responsive organic nanowire by a site-specific chemical reaction with an aromatic amine to form an azo dye structure on the surface. The resulting azo-M13-phage nanowire exhibits reversible photo-responsive properties due to the photo-switchable cis-trans isomerisation of the azo unit formed on the phage. This result shows that site-specific display of a peptide on bio-nanostructures through site-directed genetic mutagenesis can be translated into site-directed chemical reaction for developing advanced materials. The photo-responsive properties of the azo-M13-phage nanowires may open the door for the development of light controllable smart devices for use in non-linear optics, holography data storage, molecular antenna, and actuators.

Virus-based Photo-Responsive Nanowires Formed By Linking Site-Directed Mutagenesis and Chemical Reaction

PubMed Central

Murugesan, Murali; Abbineni, Gopal; Nimmo, Susan L.; Cao, Binrui; Mao, Chuanbin

2013-01-01

Owing to the genetic flexibility and error-free bulk production, bio-nanostructures such as filamentous phage showed great potential in materials synthesis, however, their photo-responsive behaviour is neither explored nor unveiled. Here we show M13 phage genetically engineered with tyrosine residues precisely fused to the major coat protein is converted into a photo-responsive organic nanowire by a site-specific chemical reaction with an aromatic amine to form an azo dye structure on the surface. The resulting azo-M13-phage nanowire exhibits reversible photo-responsive properties due to the photo-switchable cis-trans isomerisation of the azo unit formed on the phage. This result shows that site-specific display of a peptide on bio-nanostructures through site-directed genetic mutagenesis can be translated into site-directed chemical reaction for developing advanced materials. The photo-responsive properties of the azo-M13-phage nanowires may open the door for the development of light controllable smart devices for use in non-linear optics, holography data storage, molecular antenna, and actuators. PMID:23673356

Mutagenesis study to disrupt electrostatic interactions on the twofold symmetry interface of Escherichia coli bacterioferritin.

PubMed

Zhang, Yu; Wang, Lijun; Ardejani, Maziar S; Aris, Nur Fazlina; Li, Xun; Orner, Brendan P; Wang, Fei

2015-12-01

Ferritins and other cage proteins have been utilized as models to understand the fundamentals of protein folding and self-assembly. The bacterioferritin (BFR) from Escherichia coli, a maxi-ferritin made up of 24 subunits, was chosen as the basis for a mutagenesis study to investigate the role of electrostatic intermolecular interactions mediated through charged amino acids. Through structural and computational analyses, three charged amino acids R30, D56 and E60 which involved in an electrostatic interaction network were mutated to the opposite charge. Four mutants, R30D, D56R, E60H and D56R-E60H, were expressed, purified and characterized. All of the mutants fold into α-helical structures. Consistent with the computational prediction, they all show a lowered thermostability; double mutant D56R-E60H was found to be 16°C less stable than the wild type. Except for the mutant E60H, all the other mutations completely shut down the formation of protein cages to favour the dimer state in solution. The mutants, however, retain their ability to form cage-like nanostructures in the dried, surface immobilized conditions of transmission electron microscopy. Our findings confirm that even a single charge-inversion mutation at the 2-fold interface of BFR can affect the quaternary structure of its dimers and their ability to self-assemble into cage structures. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid

PubMed Central

Aboklaish, Ali F.; Dordet-Frisoni, Emilie; Citti, Christine; Toleman, Mark A; Glass, John I.; Spiller, O. Brad

2015-01-01

While transposon mutagenesis has been successfully used for Mycoplasma spp. to disrupt and determine non-essential genes, previous attempts with Ureaplasma spp. have been unsuccessful. Using a polyethylene glycol-transformation enhancing protocol, we were able to transform three separate serovars of Ureaplasma parvum with a Tn4001-based mini-transposon plasmid containing a gentamicin resistance selection marker. Despite the large degree of homology between Ureaplasma parvum and Ureaplasma urealyticum, all attempts to transform the latter in parallel failed, with the exception of a single clinical U. urealyticum isolate. PCR probing and sequencing were used to confirm transposon insertion into the bacterial genome and identify disrupted genes. Transformation of prototype serovar 3 consistently resulted in transfer only of sequence between the mini-transposon inverted repeats, but some strains showed additional sequence transfer. Transposon insertion occurred randomly in the genome resulting in unique disruption of genes UU047, UU390, UU440, UU450, UU520, UU526, UU582 for single clones from a panel of screened clones. An intergenic insertion between genes UU187 and UU188 was also characterised. Two phenotypic alterations were observed in the mutated strains: Disruption of a DEAD-box RNA helicase (UU582) altered growth kinetics, while the U. urealyticum strain lost resistance to serum attack coincident with disruption of gene UUR10_137 and loss of expression of a 41 kDa protein. Transposon mutagenesis was used successfully to insert single copies of a mini-transposon into the genome and disrupt genes leading to phenotypic changes in Ureaplasma parvum strains. This method can now be used to deliver exogenous genes for expression and determine essential genes for Ureaplasma parvum replication in culture and experimental models. PMID:25444567

Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid.

PubMed

Aboklaish, Ali F; Dordet-Frisoni, Emilie; Citti, Christine; Toleman, Mark A; Glass, John I; Spiller, O Brad

2014-11-01

While transposon mutagenesis has been successfully used for Mycoplasma spp. to disrupt and determine non-essential genes, previous attempts with Ureaplasma spp. have been unsuccessful. Using a polyethylene glycol-transformation enhancing protocol, we were able to transform three separate serovars of Ureaplasma parvum with a Tn4001-based mini-transposon plasmid containing a gentamicin resistance selection marker. Despite the large degree of homology between Ureaplasma parvum and Ureaplasma urealyticum, all attempts to transform the latter in parallel failed, with the exception of a single clinical U. urealyticum isolate. PCR probing and sequencing were used to confirm transposon insertion into the bacterial genome and identify disrupted genes. Transformation of prototype serovar 3 consistently resulted in transfer only of sequence between the mini-transposon inverted repeats, but some strains showed additional sequence transfer. Transposon insertion occurred randomly in the genome resulting in unique disruption of genes UU047, UU390, UU440, UU450, UU520, UU526, UU582 for single clones from a panel of screened clones. An intergenic insertion between genes UU187 and UU188 was also characterised. Two phenotypic alterations were observed in the mutated strains: Disruption of a DEAD-box RNA helicase (UU582) altered growth kinetics, while the U. urealyticum strain lost resistance to serum attack coincident with disruption of gene UUR10_137 and loss of expression of a 41 kDa protein. Transposon mutagenesis was used successfully to insert single copies of a mini-transposon into the genome and disrupt genes leading to phenotypic changes in Ureaplasma parvum strains. This method can now be used to deliver exogenous genes for expression and determine essential genes for Ureaplasma parvum replication in culture and experimental models. Copyright © 2014 Elsevier GmbH. All rights reserved.

Prediction of hot spot residues at protein-protein interfaces by combining machine learning and energy-based methods.

PubMed

Lise, Stefano; Archambeau, Cedric; Pontil, Massimiliano; Jones, David T

2009-10-30

Alanine scanning mutagenesis is a powerful experimental methodology for investigating the structural and energetic characteristics of protein complexes. Individual amino-acids are systematically mutated to alanine and changes in free energy of binding (DeltaDeltaG) measured. Several experiments have shown that protein-protein interactions are critically dependent on just a few residues ("hot spots") at the interface. Hot spots make a dominant contribution to the free energy of binding and if mutated they can disrupt the interaction. As mutagenesis studies require significant experimental efforts, there is a need for accurate and reliable computational methods. Such methods would also add to our understanding of the determinants of affinity and specificity in protein-protein recognition. We present a novel computational strategy to identify hot spot residues, given the structure of a complex. We consider the basic energetic terms that contribute to hot spot interactions, i.e. van der Waals potentials, solvation energy, hydrogen bonds and Coulomb electrostatics. We treat them as input features and use machine learning algorithms such as Support Vector Machines and Gaussian Processes to optimally combine and integrate them, based on a set of training examples of alanine mutations. We show that our approach is effective in predicting hot spots and it compares favourably to other available methods. In particular we find the best performances using Transductive Support Vector Machines, a semi-supervised learning scheme. When hot spots are defined as those residues for which DeltaDeltaG >or= 2 kcal/mol, our method achieves a precision and a recall respectively of 56% and 65%. We have developed an hybrid scheme in which energy terms are used as input features of machine learning models. This strategy combines the strengths of machine learning and energy-based methods. Although so far these two types of approaches have mainly been applied separately to biomolecular problems

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

PubMed

Tincher, Clayton; Long, Hongan; Behringer, Megan; Walker, Noah; Lynch, Michael

2017-10-05

Mutations induced by pollutants may promote pathogen evolution, for example by accelerating mutations conferring antibiotic resistance. Generally, evaluating the genome-wide mutagenic effects of long-term sublethal pollutant exposure at single-nucleotide resolution is extremely difficult. To overcome this technical barrier, we use the mutation accumulation/whole-genome sequencing (MA/WGS) method as a mutagenicity test, to quantitatively evaluate genome-wide mutagenesis of Escherichia coli after long-term exposure to a wide gradient of the glyphosate-based herbicide (GBH) Roundup Concentrate Plus. The genome-wide mutation rate decreases as GBH concentration increases, suggesting that even long-term GBH exposure does not compromise the genome stability of bacteria. Copyright © 2017 Tincher et al.

Transcription factor-based modulation of neural stem cell differentiation using direct protein transduction

PubMed Central

Stock, Kristin; Nolden, Lars; Edenhofer, Frank; Quandel, Tamara

2010-01-01

In contrast to conventional gene transfer strategies, the direct introduction of recombinant proteins into cells bypasses the risk of insertional mutagenesis and offers an alternative to genetic intervention. Here, we explore whether protein transduction of the gliogenic transcription factor Nkx2.2 can be used to promote oligodendroglial differentiation of mouse embryonic stem cell (ESC)-derived neural stem cells (NSC). To that end, a recombinant cell-permeant form of Nkx2.2 protein was generated. Exposure of ESC-derived NSC to the recombinant protein and initiation of differentiation resulted in a two-fold increase in the number of oligodendrocytes. Furthermore, Nkx2.2-transduced cells exhibited a more mature oligodendroglial phenotype. Comparative viral gene transfer studies showed that the biological effect of Nkx2.2 protein transduction is comparable to that obtained by lentiviral transduction. The results of this proof-of-concept study depict direct intracellular delivery of transcription factors as alternative modality to control lineage differentiation in NSC cultures without genetic modification. Electronic supplementary material The online version of this article (doi:10.1007/s00018-010-0347-1) contains supplementary material, which is available to authorized users. PMID:20352468

Thermostabilisation of membrane proteins for structural studies

PubMed Central

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.

2017-01-01

The thermostability of an integral membrane protein in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals suitable for structure determination. However, many mammalian membrane proteins are too unstable for crystallisation. We developed a thermostabilisation strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes approximately 6-12 months to thermostabilise a G protein-coupled receptor (GPCR) containing 300 amino acid residues. The resulting thermostabilised membrane proteins are more easily crystallised 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 thermostabilised receptors bound to low affinity ligands. Protocols and advice are given on how to develop thermostability assays for membrane proteins and how to combine mutations to make an optimally stable mutant suitable for structural studies. PMID:27466713

Structural interpretation of P2X receptor mutagenesis studies on drug action

PubMed Central

Evans, Richard J

2010-01-01

P2X receptors for ATP are ligand gated cation channels that form from the trimeric assembly of subunits with two transmembrane segments, a large extracellular ligand binding loop, and intracellular amino and carboxy termini. The receptors are expressed throughout the body, involved in functions ranging from blood clotting to inflammation, and may provide important targets for novel therapeutics. Mutagenesis based studies have been used to develop an understanding of the molecular basis of their pharmacology with the aim of developing models of the ligand binding site. A crystal structure for the zebra fish P2X4 receptor in the closed agonist unbound state has been published recently, which provides a major advance in our understanding of the receptors. This review gives an overview of mutagenesis studies that have led to the development of a model of the ATP binding site, as well as identifying residues contributing to allosteric regulation and antagonism. These studies are discussed with reference to the crystal to provide a structural interpretation of the molecular basis of drug action. PMID:20977449

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

PubMed

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

2014-06-01

Recent computational and bioinformatics advances have enabled the efficient creation of novel biocatalysts by reducing amino acid variability at hot spot regions. To further expand the utility of this strategy, we present here a tool called Multi-site Degenerate Codon Analyzer (MDC-Analyzer) for the automated design of intelligent mutagenesis libraries that can completely cover user-defined randomized sequences, especially when multiple contiguous and/or adjacent sites are targeted. By initially defining an objective function, the possible optimal degenerate PCR primer profiles could be automatically explored using the heuristic approach of Greedy Best-First-Search. Compared to the previously developed DC-Analyzer, MDC-Analyzer allows for the existence of a small amount of undesired sequences as a tradeoff between the number of degenerate primers and the encoded library size while still providing all the benefits of DC-Analyzer with the ability to randomize multiple contiguous sites. MDC-Analyzer was validated using a series of randomly generated mutation schemes and experimental case studies on the evolution of halohydrin dehalogenase, which proved that the MDC methodology is more efficient than other methods and is particularly well-suited to exploring the sequence space of proteins using data-driven protein engineering strategies.

The Structure of Urease Activiation Complexes Examined by Flexibility Analysis, Mutagenesis, and Small-angle X-ray Scattering Approaches

DOE Office of Scientific and Technical Information (OSTI.GOV)

Quiroz, Soledad; Sukuru, Sai Chetan K.; Hausinger, Robert P.

2008-01-01

Conformational changes of Klebsiella aerogenes urease apoprotein (UreABC){sub 3} induced upon binding of the UreD and UreF accessory proteins were examined by a combination of flexibility analysis, mutagenesis, and small-angle X-ray scattering (SAXS). ProFlex analysis of urease provided evidence that the major domain of UreB can move in a hinge-like motion to account for prior chemical cross-linking results. Rigidification of the UreB hinge region, accomplished through a G11P mutation, reduced the extent of urease activation, in part by decreasing the nickel content of the mutant enzyme, and by sequestering a portion of the urease apoprotein in a novel activation complexmore » that includes all of the accessory proteins. SAXS analyses of urease, (UreABC-UreD){sub 3}, and (UreABC-UreDF){sub 3} confirm that UreD and UreF bind near UreB at the periphery of the (UreAC){sub 3} structure. This study supports an activation model in which a domain-shifted UreB conformation in (UreABC-UreDF){sub 3} allows CO{sub 2} and nickel ions to gain access to the nascent active site.« less

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

PubMed

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

2016-01-01

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

Heavy ion mutagenesis combined with triclosan screening provides a new strategy for improving the arachidonic acid yield in Mortierella alpina.

PubMed

Zhang, Huidan; Lu, Dong; Li, Xin; Feng, Yingang; Cui, Qiu; Song, Xiaojin

2018-05-02

Arachidonic acid (ARA), which is a ω-6 polyunsaturated fatty acid, has a wide range of biological activities and is an essential component of cellular membranes in some human tissues. Mortierella alpina is the best strain for industrial production of ARA. To increase its yield of arachidonic acid, heavy ion beam irradiation mutagenesis of Mortierella alpina was carried out in combination with triclosan and octyl gallate treatment. The obtained mutant strain F-23 ultimately achieved an ARA yield of 5.26 g L - 1 , which is 3.24 times higher than that of the wild-type strain. In addition, quantitative real-time PCR confirmed that the expression levels of fatty acid synthase (FAS), Δ5-desaturase, Δ6-desaturase, and Δ9-desaturase were all significantly up-regulated in the mutant F-23 strain, especially Δ6- and Δ9-desaturase, which were up-regulated 3- and 2-fold, respectively. This study confirmed a feasible mutagenesis breeding strategy for improving ARA production and provided a mutant of Mortierella alpina with high ARA yield.

Oxidative mutagenesis of doxorubicin-Fe(III) complex.

PubMed

Kostoryz, E L; Yourtee, D M

2001-02-20

Doxorubicin has a high affinity for inorganic iron, Fe(III), and has potential to form doxorubicin-Fe(III) complexes in biological systems. Indirect involvement of iron has been substantiated in the oxidative mutagenicity of doxorubicin. In this study, however, direct involvement of Fe(III) was evaluated in mutagenicity studies with the doxorubicin-Fe(III) complex. The Salmonella mutagenicity assay with strain TA102 was used with a pre-incubation step. The highest mutagenicity of doxorubicin-Fe(III) complex was observed at the dose of 2.5nmol/plate of the complex. The S9-mix decreased this highest mutagenicity but increased the number of revertants at a higher dose of 10nmol/plate of the complex. On the other hand, the mutagenicity of the doxorubicin-Fe(III) complex at the doses of 0.25, 0.5, 1 and 2nmol/plate was enhanced about twice by the addition of glutathione plus H(2)O(2). This enhanced mutagenicity as well as of the complex itself, the complex plus glutathione, and the complex plus H(2)O(2) were reduced by the addition of ADR-529, an Fe(III) chelator, and potassium iodide, a hydroxyl radical scavenger. These results indicate that doxorubicin-Fe(III) complex exert the mutagenicity through oxidative DNA damage and that Fe(III) is a required element in the mutagenesis of doxorubicin.

MMTV insertional mutagenesis identifies genes, gene families and pathways involved in mammary cancer.

PubMed

Theodorou, Vassiliki; Kimm, Melanie A; Boer, Mandy; Wessels, Lodewyk; Theelen, Wendy; Jonkers, Jos; Hilkens, John

2007-06-01

We performed a high-throughput retroviral insertional mutagenesis screen in mouse mammary tumor virus (MMTV)-induced mammary tumors and identified 33 common insertion sites, of which 17 genes were previously not known to be associated with mammary cancer and 13 had not previously been linked to cancer in general. Although members of the Wnt and fibroblast growth factors (Fgf) families were frequently tagged, our exhaustive screening for MMTV insertion sites uncovered a new repertoire of candidate breast cancer oncogenes. We validated one of these genes, Rspo3, as an oncogene by overexpression in a p53-deficient mammary epithelial cell line. The human orthologs of the candidate oncogenes were frequently deregulated in human breast cancers and associated with several tumor parameters. Computational analysis of all MMTV-tagged genes uncovered specific gene families not previously associated with cancer and showed a significant overrepresentation of protein domains and signaling pathways mainly associated with development and growth factor signaling. Comparison of all tagged genes in MMTV and Moloney murine leukemia virus-induced malignancies showed that both viruses target mostly different genes that act predominantly in distinct pathways.

Biologically active LIL proteins built with minimal chemical diversity

PubMed Central

Heim, Erin N.; Marston, Jez L.; Federman, Ross S.; Edwards, Anne P. B.; Karabadzhak, Alexander G.; Petti, Lisa M.; Engelman, Donald M.; DiMaio, Daniel

2015-01-01

We have constructed 26-amino acid transmembrane proteins that specifically transform cells but consist of only two different amino acids. Most proteins are long polymers of amino acids with 20 or more chemically distinct side-chains. The artificial transmembrane proteins reported here are the simplest known proteins with specific biological activity, consisting solely of an initiating methionine followed by specific sequences of leucines and isoleucines, two hydrophobic amino acids that differ only by the position of a methyl group. We designate these proteins containing leucine (L) and isoleucine (I) as LIL proteins. These proteins functionally interact with the transmembrane domain of the platelet-derived growth factor β-receptor and specifically activate the receptor to transform cells. Complete mutagenesis of these proteins identified individual amino acids required for activity, and a protein consisting solely of leucines, except for a single isoleucine at a particular position, transformed cells. These surprisingly simple proteins define the minimal chemical diversity sufficient to construct proteins with specific biological activity and change our view of what can constitute an active protein in a cellular context. PMID:26261320

A protocol for chemical mutagenesis in Strongyloides ratti.

PubMed

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

2015-11-01

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

Identification of a halotolerant mutant via in vitro mutagenesis in the cyanobacterium Fremyella diplosiphon

USDA-ARS?s Scientific Manuscript database

Energy metabolism and photosynthetic pigment accumulation are affected by salt stress in cyanobacteria leading to cessation of growth. The effect of salinity on the fresh water cyanobacteria, Fremyella diplosiphon was investigated and mutagenesis-based efforts were undertaken to enhance salt toleran...

Increasing Protein Charge State When Using Laser Electrospray Mass Spectrometry

NASA Astrophysics Data System (ADS)

Karki, Santosh; Flanigan, Paul M.; Perez, Johnny J.; Archer, Jieutonne J.; Levis, Robert J.

2015-05-01

Femtosecond (fs) laser vaporization is used to transfer cytochrome c, myoglobin, lysozyme, and ubiquitin from the condensed phase into an electrospray (ES) plume consisting of a mixture of a supercharging reagent, m-nitrobenzyl alcohol ( m-NBA), and trifluoroacetic acid (TFA), acetic acid (AA), or formic acid (FA). Interaction of acid-sensitive proteins like cytochrome c and myoglobin with the highly charged ES droplets resulted in a shift to higher charge states in comparison with acid-stable proteins like lysozyme and ubiquitin. Laser electrospray mass spectrometry (LEMS) measurements showed an increase in both the average charge states (Zavg) and the charge state with maximum intensity (Zmode) for acid-sensitive proteins compared with conventional electrospray ionization mass spectrometry (ESI-MS) under equivalent solvent conditions. A marked increase in ion abundance of higher charge states was observed for LEMS in comparison with conventional electrospray for cytochrome c (ranging from 19+ to 21+ versus 13+ to 16+) and myoglobin (ranging from 19+ to 26+ versus 18+ to 21+) using an ES solution containing m-NBA and TFA. LEMS measurements as a function of electrospray flow rate yielded increasing charge states with decreasing flow rates for cytochrome c and myoglobin.

Molecular characterization of the staphylococcal multidrug resistance export protein QacC.

PubMed Central

Paulsen, I T; Brown, M H; Dunstan, S J; Skurray, R A

1995-01-01

The QacC polypeptide is a member of a family of small membrane proteins which confer resistance to toxic compounds. The staphylococcal qacC gene confers resistance to toxic organic cations via proton-dependent export. The membrane topology of the QacC polypeptide was investigated by constructing and analyzing a series of qacC-phoA and qacC-lacZ fusions. From these analyses, most of the predicted features of the QacC protein were verified, although data regarding the possible orientation of the COOH region were not conclusive. The role of the sole cysteine residue, Cys-42, in QacC was studied by using the sulfhydryl reagent N-ethylmaleimide and site-directed mutagenesis. N-Ethylmaleimide was shown to inhibit qacC-mediated ethidium export. Multiple amino acid substitutions were made for Cys-42, and mutations at this location had various effects on resistance specificity. This suggests that the Cys-42 residue may be located near a region of QacC that is involved in substrate recognition. Mutagenesis of conserved residues in QacC indicated that Tyr-59 and Trp-62 also play an essential structural or functional role in QacC. PMID:7751293

Multiplex detection of protein-protein interactions using a next generation luciferase reporter.

PubMed

Verhoef, Lisette G G C; Mattioli, Michela; Ricci, Fernanda; Li, Yao-Cheng; Wade, Mark

2016-02-01

Cell-based assays of protein-protein interactions (PPIs) using split reporter proteins can be used to identify PPI agonists and antagonists. Generally, such assays measure one PPI at a time, and thus counterscreens for on-target activity must be run in parallel or at a subsequent stage; this increases both the cost and time during screening. Split luciferase systems offer advantages over those that use split fluorescent proteins (FPs). This is since split luciferase offers a greater signal:noise ratio and, unlike split FPs, the PPI can be reversed upon small molecule treatment. While multiplexed PPI assays using luciferase have been reported, they suffer from low signal:noise and require fairly complex spectral deconvolution during analysis. Furthermore, the luciferase enzymes used are large, which limits the range of PPIs that can be interrogated due to steric hindrance from the split luciferase fragments. Here, we report a multiplexed PPI assay based on split luciferases from Photinus pyralis (firefly luciferase, FLUC) and the deep-sea shrimp, Oplophorus gracilirostris (NanoLuc, NLUC). Specifically, we show that the binding of the p53 tumor suppressor to its two major negative regulators, MDM2 and MDM4, can be simultaneously measured within the same sample, without the requirement for complex filters or deconvolution. We provide chemical and genetic validation of this system using MDM2-targeted small molecules and mutagenesis, respectively. Combined with the superior signal:noise and smaller size of split NanoLuc, this multiplexed PPI assay format can be exploited to study the induction or disruption of pairwise interactions that are prominent in many cell signaling pathways. Copyright © 2015 Elsevier B.V. All rights reserved.