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Sample records for tn5051-like mer-containing transposon

  1. A Tn5051-like mer-containing transposon identified in a heavy metal tolerant strain Achromobacter sp. AO22

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    Bhave Mrinal

    2009-03-01

    Full Text Available Abstract Background Achromobacter sp. AO22 (formerly Alcaligenes sp. AO22, a bacterial strain isolated from a lead-contaminated industrial site in Australia, was previously found to be resistant to moderate to high levels of mercury, copper and other heavy metals. However, the nature and location of the genetic basis for mercuric ion resistance in this strain, had not been previously identified. Findings Achromobacter sp. AO22 contains a functional mer operon with all four essential genes (merRTPA and shows >99% DNA sequence identity to that of Tn501. The mer operon was present on a transposon, designated TnAO22, captured by introducing a broad-host-range IncP plasmid into Achromobacter sp. AO22 and subsequently transferring it to E. coli recipients. The transposition frequency of TnAO22 was 10-2 to 10-3 per target plasmid transferred. Analysis of TnAO22 sequence revealed it belonged to the Tn21 subgroup of the Tn3 superfamily of transposons, with the transposition module having >99% identity with Tn5051 of a Pseudomonas putida strain isolated from a water sample in New York. Conclusion TnAO22 is thus a new variant of Tn5051 of the Tn3 superfamily and the transposon and its associated mercury resistance system are among the few such systems reported in a soil bacterium. Achromobacter sp. AO22 can thus be exploited for applications such as in situ mercury bioremediation of contaminated sites, or the mobile unit and mer operon could be mobilized to other bacteria for similar purposes.

  2. Phage transposon mutagenesis.

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    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.

  3. Global mapping of transposon location.

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    Abram Gabriel

    2006-12-01

    Full Text Available Transposable genetic elements are ubiquitous, yet their presence or absence at any given position within a genome can vary between individual cells, tissues, or strains. Transposable elements have profound impacts on host genomes by altering gene expression, assisting in genomic rearrangements, causing insertional mutations, and serving as sources of phenotypic variation. Characterizing a genome's full complement of transposons requires whole genome sequencing, precluding simple studies of the impact of transposition on interindividual variation. Here, we describe a global mapping approach for identifying transposon locations in any genome, using a combination of transposon-specific DNA extraction and microarray-based comparative hybridization analysis. We use this approach to map the repertoire of endogenous transposons in different laboratory strains of Saccharomyces cerevisiae and demonstrate that transposons are a source of extensive genomic variation. We also apply this method to mapping bacterial transposon insertion sites in a yeast genomic library. This unique whole genome view of transposon location will facilitate our exploration of transposon dynamics, as well as defining bases for individual differences and adaptive potential.

  4. Mariner and the ITm Superfamily of Transposons.

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    Tellier, Michael; Bouuaert, Corentin Claeys; Chalmers, Ronald

    2015-04-01

    The IS630-Tc1-mariner (ITm) family of transposons is one of the most widespread in nature. The phylogenetic distribution of its members shows that they do not persist for long in a given lineage, but rely on frequent horizontal transfer to new hosts. Although they are primarily selfish genomic-parasites, ITm transposons contribute to the evolution of their hosts because they generate variation and contribute protein domains and regulatory regions. Here we review the molecular mechanism of ITm transposition and its regulation. We focus mostly on the mariner elements, which are understood in the greatest detail owing to in vitro reconstitution and structural analysis. Nevertheless, the most important characteristics are probably shared across the grouping. Members of the ITm family are mobilized by a cut-and-paste mechanism and integrate at 5'-TA dinucleotide target sites. The elements encode a single transposase protein with an N-terminal DNA-binding domain and a C-terminal catalytic domain. The phosphoryl-transferase reactions during the DNA-strand breaking and joining reactions are performed by the two metal-ion mechanism. The metal ions are coordinated by three or four acidic amino acid residues located within an RNase H-like structural fold. Although all of the strand breaking and joining events at a given transposon end are performed by a single molecule of transposase, the reaction is coordinated by close communication between transpososome components. During transpososome assembly, transposase dimers compete for free transposon ends. This helps to protect the host by dampening an otherwise exponential increase in the rate of transposition as the copy number increases.

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

    DEFF Research Database (Denmark)

    Nilsson, Martin; Christiansen, Natalia; Høiby, Niels

    2014-01-01

    This article describes the construction and characterization of a mariner-based transposon vector designed for use in oral streptococci, but with a potential use in other Gram-positive bacteria. The new transposon vector, termed pMN100, contains the temperature-sensitive origin of replication rep...... 5000 mutants was used in a screen to identify genes involved in the production of sucrose-dependent extracellular matrix components. Mutants with transposon inserts in genes encoding glycosyltransferases and the competence-related secretory locus were predominantly found in this screen....

  6. Transposon display supports transpositional activity of P elements in ...

    Indian Academy of Sciences (India)

    . Abstract. Mobilization of two P element subfamilies (canonical and O-type) from Drosophila sturtevanti and D. saltans was evaluated for copy number and transposition activity using the transposon display (TD) technique. Pairwise distances ...

  7. Gene-specific cell labeling using MiMIC transposons.

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    Gnerer, Joshua P; Venken, Koen J T; Dierick, Herman A

    2015-04-30

    Binary expression systems such as GAL4/UAS, LexA/LexAop and QF/QUAS have greatly enhanced the power of Drosophila as a model organism by allowing spatio-temporal manipulation of gene function as well as cell and neural circuit function. Tissue-specific expression of these heterologous transcription factors relies on random transposon integration near enhancers or promoters that drive the binary transcription factor embedded in the transposon. Alternatively, gene-specific promoter elements are directly fused to the binary factor within the transposon followed by random or site-specific integration. However, such insertions do not consistently recapitulate endogenous expression. We used Minos-Mediated Integration Cassette (MiMIC) transposons to convert host loci into reliable gene-specific binary effectors. MiMIC transposons allow recombinase-mediated cassette exchange to modify the transposon content. We developed novel exchange cassettes to convert coding intronic MiMIC insertions into gene-specific binary factor protein-traps. In addition, we expanded the set of binary factor exchange cassettes available for non-coding intronic MiMIC insertions. We show that binary factor conversions of different insertions in the same locus have indistinguishable expression patterns, suggesting that they reliably reflect endogenous gene expression. We show the efficacy and broad applicability of these new tools by dissecting the cellular expression patterns of the Drosophila serotonin receptor gene family. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Random insertion and gene disruption via transposon mutagenesis of Ureaplasma parvum using a mini-transposon plasmid.

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    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.

  9. Enzymatic engineering of the porcine genome with transposons and recombinases

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    Carlson Daniel F

    2007-07-01

    Full Text Available Abstract Background Swine is an important agricultural commodity and biomedical model. Manipulation of the pig genome provides opportunity to improve production efficiency, enhance disease resistance, and add value to swine products. Genetic engineering can also expand the utility of pigs for modeling human disease, developing clinical treatment methodologies, or donating tissues for xenotransplantation. Realizing the full potential of pig genetic engineering requires translation of the complete repertoire of genetic tools currently employed in smaller model organisms to practical use in pigs. Results Application of transposon and recombinase technologies for manipulation of the swine genome requires characterization of their activity in pig cells. We tested four transposon systems- Sleeping Beauty, Tol2, piggyBac, and Passport in cultured porcine cells. Transposons increased the efficiency of DNA integration up to 28-fold above background and provided for precise delivery of 1 to 15 transgenes per cell. Both Cre and Flp recombinase were functional in pig cells as measured by their ability to remove a positive-negative selection cassette from 16 independent clones and over 20 independent genomic locations. We also demonstrated a Cre-dependent genetic switch capable of eliminating an intervening positive-negative selection cassette and activating GFP expression from episomal and genome-resident transposons. Conclusion We have demonstrated for the first time that transposons and recombinases are capable of mobilizing DNA into and out of the porcine genome in a precise and efficient manner. This study provides the basis for developing transposon and recombinase based tools for genetic engineering of the swine genome.

  10. Towards controlled mutagenesis with transposons Ac and Tam3

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    Haring, M; Veken, J; Windrich, R; Kneppers, T; Rommens, C; Nijkamp, H J.J.; Hille, J [Department of Genetics, Free University, Amsterdam (Netherlands)

    1990-01-01

    Full text: The discovery of mobile genetic elements in plants has permitted the use of these transposons for insertional mutagenesis. This applies so far only to Zea mays and Antirrhinum majus, because other plant transposable elements have not been characterised so thoroughly at the genetic and the molecular level. To establish whether transposons (Ac from maize and Tam3 from Antirrhinum) remain mobile in heterologous hosts, either in somatic tissue or after meiosis, a phenotypic assay system for transposition was developed. The separation of the two transposition functions will allow controlled mutagenesis of plant genes. Our results indicate that both transposable elements remain active in heterologous hosts. (author)

  11. Himar1 Transposon for Efficient Random Mutagenesis in Aggregatibacter actinomycetemcomitans

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    Qinfeng Ding

    2017-09-01

    Full Text Available 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.

  12. Identification and evolutionary dynamics of cacta DNA transposons in brassica

    International Nuclear Information System (INIS)

    Nouroz, F.; Noreen, S.; Harrison, J.S.H.

    2017-01-01

    Transposable elements are the major drivers of genome evolution and plasticity. Due to their transposition mode, they are classified into two major classes as Retrotransposons and DNA transposons. The En/Spm or CACTA elements are diverse group of DNA transposons proliferating in plant genomes. Various bioinformatics and molecular approaches were used for identification and distribution of CACTA transposons in Brassica genome. A combination of dot plot analysis and BLASTN searches yielded 35 autonomous and 7 non-autonomous CACTA elements in Brassica. The elements ranged in sizes from 1.2 kb non-autonomous elements to 11kb autonomous elements, terminated by 3 bp Target Site Duplication (TSD) and ~15 bp conserved Terminal Inverted Repeat (TIR) motifs (5'-CACTACAAGAAAACA-3'), with heterogeneous internal regions. The transposase (TNP) was identified from autonomous CACTA elements, while other protein domains from Brassica and other plants CACTA revealed similar organizations with minor differences. Both transposases (TNPD, TNPA) are present in most CACTA, while a few CACTA harboured an additional ATHILA ORF1-like domain. The PCR analysis amplified the CACTA transposases from 40 Brassica accessions (A, B, and C-genome) suggesting their distribution among various Brassica crops. A detailed characterization and evolutionary analysis of the identified CACTA elements allowed some to be placed in genome-specific groups, while most of them (Brassica-Arabidopsis elements) have followed the same evolutionary line. The distribution of CACTA in Brassica concluded that 3 bp TSDs generating CACTA transposons contributed significantly to genome size and evolution of Brassica genome. (author)

  13. Transposon domestication versus mutualism in ciliate genome rearrangements.

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    Alexander Vogt

    Full Text Available Ciliated protists rearrange their genomes dramatically during nuclear development via chromosome fragmentation and DNA deletion to produce a trimmer and highly reorganized somatic genome. The deleted portion of the genome includes potentially active transposons or transposon-like sequences that reside in the germline. Three independent studies recently showed that transposase proteins of the DDE/DDD superfamily are indispensible for DNA processing in three distantly related ciliates. In the spirotrich Oxytricha trifallax, high copy-number germline-limited transposons mediate their own excision from the somatic genome but also contribute to programmed genome rearrangement through a remarkable transposon mutualism with the host. By contrast, the genomes of two oligohymenophorean ciliates, Tetrahymena thermophila and Paramecium tetraurelia, encode homologous PiggyBac-like transposases as single-copy genes in both their germline and somatic genomes. These domesticated transposases are essential for deletion of thousands of different internal sequences in these species. This review contrasts the events underlying somatic genome reduction in three different ciliates and considers their evolutionary origins and the relationships among their distinct mechanisms for genome remodeling.

  14. Transposons As Tools for Functional Genomics in Vertebrate Models.

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    Kawakami, Koichi; Largaespada, David A; Ivics, Zoltán

    2017-11-01

    Genetic tools and mutagenesis strategies based on transposable elements are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of their inherent capacity to insert into DNA, transposons can be developed into powerful tools for chromosomal manipulations. Transposon-based forward mutagenesis screens have numerous advantages including high throughput, easy identification of mutated alleles, and providing insight into genetic networks and pathways based on phenotypes. For example, the Sleeping Beauty transposon has become highly instrumental to induce tumors in experimental animals in a tissue-specific manner with the aim of uncovering the genetic basis of diverse cancers. Here, we describe a battery of mutagenic cassettes that can be applied in conjunction with transposon vectors to mutagenize genes, and highlight versatile experimental strategies for the generation of engineered chromosomes for loss-of-function as well as gain-of-function mutagenesis for functional gene annotation in vertebrate models, including zebrafish, mice, and rats. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Epigenetics and Evolution: Transposons and the Stochastic Epigenetic Modification Model

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    Sergio Branciamore

    2015-04-01

    Full Text Available In addition to genetic variation, epigenetic variation and transposons can greatly affect the evolutionary fitnesses landscape and gene expression. Previously we proposed a mathematical treatment of a general epigenetic variation model that we called Stochastic Epigenetic Modification (SEM model. In this study we follow up with a special case, the Transposon Silencing Model (TSM, with, once again, emphasis on quantitative treatment. We have investigated the evolutionary effects of epigenetic changes due to transposon (T insertions; in particular, we have considered a typical gene locus A and postulated that (i the expression level of gene A depends on the epigenetic state (active or inactive of a cis- located transposon element T, (ii stochastic variability in the epigenetic silencing of T occurs only in a short window of opportunity during development, (iii the epigenetic state is then stable during further development, and (iv the epigenetic memory is fully reset at each generation. We develop the model using two complementary approaches: a standard analytical population genetics framework (di usion equations and Monte-Carlo simulations. Both approaches led to similar estimates for the probability of fixation and time of fixation of locus TA with initial frequency P in a randomly mating diploid population of effective size Ne. We have ascertained the e ect that ρ, the probability of transposon Modification during the developmental window, has on the population (species. One of our principal conclusions is that as ρ increases, the pattern of fixation of the combined TA locus goes from "neutral" to "dominant" to "over-dominant". We observe that, under realistic values of ρ, epigenetic Modifications can provide an e cient mechanism for more rapid fixation of transposons and cis-located gene alleles. The results obtained suggest that epigenetic silencing, even if strictly transient (being reset at each generation, can still have signi cant

  16. Remobilization of Sleeping Beauty transposons in the germline of Xenopus tropicalis

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    Yergeau Donald A

    2011-11-01

    Full Text Available Abstract Background The Sleeping Beauty (SB transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization. Results Transgenic frogs that express the SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'. Conclusions In this study, we demonstrate that SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.

  17. Transposon-mediated BAC transgenesis in zebrafish and mice

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    Sumiyama Kenta

    2009-10-01

    Full Text Available Abstract Background Bacterial artificial chromosomes (BACs are among the most widely used tools for studies of gene regulation and function in model vertebrates, yet methods for predictable delivery of BAC transgenes to the genome are currently limited. This is because BAC transgenes are usually microinjected as naked DNA into fertilized eggs and are known to integrate as multi-copy concatamers in the genome. Although conventional methods for BAC transgenesis have been very fruitful, complementary methods for generating single copy BAC integrations would be desirable for many applications. Results We took advantage of the precise cut-and-paste behavior of a natural transposon, Tol2, to develop a new method for BAC transgenesis. In this new method, the minimal sequences of the Tol2 transposon were used to deliver precisely single copies of a ~70 kb BAC transgene to the zebrafish and mouse genomes. We mapped the BAC insertion sites in the genome by standard PCR methods and confirmed transposase-mediated integrations. Conclusion The Tol2 transposon has a surprisingly large cargo capacity that can be harnessed for BAC transgenesis. The precise delivery of single-copy BAC transgenes by Tol2 represents a useful complement to conventional BAC transgenesis, and could aid greatly in the production of transgenic fish and mice for genomics projects, especially those in which single-copy integrations are desired.

  18. Transposon mutagenesis in Bifidobacterium breve: construction and characterization of a Tn5 transposon mutant library for Bifidobacterium breve UCC2003.

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    Ruiz, Lorena; Motherway, Mary O'Connell; Lanigan, Noreen; van Sinderen, Douwe

    2013-01-01

    Bifidobacteria are claimed to contribute positively to human health through a range of beneficial or probiotic activities, including amelioration of gastrointestinal and metabolic disorders, and therefore this particular group of gastrointestinal commensals has enjoyed increasing industrial and scientific attention in recent years. However, the molecular mechanisms underlying these probiotic mechanisms are still largely unknown, mainly due to the fact that molecular tools for bifidobacteria are rather poorly developed, with many strains lacking genetic accessibility. In this work, we describe the generation of transposon insertion mutants in two bifidobacterial strains, B. breve UCC2003 and B. breve NCFB2258. We also report the creation of the first transposon mutant library in a bifidobacterial strain, employing B. breve UCC2003 and a Tn5-based transposome strategy. The library was found to be composed of clones containing single transposon insertions which appear to be randomly distributed along the genome. The usefulness of the library to perform phenotypic screenings was confirmed through identification and analysis of mutants defective in D-galactose, D-lactose or pullulan utilization abilities.

  19. Transposon mutagenesis in Bifidobacterium breve: construction and characterization of a Tn5 transposon mutant library for Bifidobacterium breve UCC2003.

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    Lorena Ruiz

    Full Text Available Bifidobacteria are claimed to contribute positively to human health through a range of beneficial or probiotic activities, including amelioration of gastrointestinal and metabolic disorders, and therefore this particular group of gastrointestinal commensals has enjoyed increasing industrial and scientific attention in recent years. However, the molecular mechanisms underlying these probiotic mechanisms are still largely unknown, mainly due to the fact that molecular tools for bifidobacteria are rather poorly developed, with many strains lacking genetic accessibility. In this work, we describe the generation of transposon insertion mutants in two bifidobacterial strains, B. breve UCC2003 and B. breve NCFB2258. We also report the creation of the first transposon mutant library in a bifidobacterial strain, employing B. breve UCC2003 and a Tn5-based transposome strategy. The library was found to be composed of clones containing single transposon insertions which appear to be randomly distributed along the genome. The usefulness of the library to perform phenotypic screenings was confirmed through identification and analysis of mutants defective in D-galactose, D-lactose or pullulan utilization abilities.

  20. [Differential expression of DTSsa4 Tc1-like transposons in closely related populations of Baikal ciscoes].

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    Bychenko, O S; Sukhanova, L V; Azhikina, T L; Sverdlov, E D

    2009-01-01

    Two representatives of Baikal ciscoes - lake cisco and omul - diverged from a common ancestor as recently as 10-20 thousand years ago. We have found an increasing expression level of DTSsa4 Tc1-like DNA transposons in cisco and omul brains. The mapping of the sequences of these transposons from Salmo salar and Danio rerio genomes has shown that in some cases, these transposons are located in the 5' and 3' regions, as well as in the promoter regions of various genes. Probably, Tc1-like transposons affect the activity of neighboring genes, providing the adaptive divergence of the cisco population.

  1. Using PATIMDB to create bacterial transposon insertion mutant libraries.

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    Urbach, Jonathan M; Wei, Tao; Liberati, Nicole; Grenfell-Lee, Daniel; Villanueva, Jacinto; Wu, Gang; Ausubel, Frederick M

    2009-04-01

    PATIMDB is a software package for facilitating the generation of transposon mutant insertion libraries. The software has two main functions: process tracking and automated sequence analysis. The process tracking function specifically includes recording the status and fates of multiwell plates and samples in various stages of library construction. Automated sequence analysis refers specifically to the pipeline of sequence analysis starting with ABI files from a sequencing facility and ending with insertion location identifications. The protocols in this unit describe installation and use of PATIMDB software.

  2. Genome-wide analysis of Tol2 transposon reintegration in zebrafish

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    Parinov Sergey

    2009-09-01

    Full Text Available Abstract Background Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome. Results We performed a large-scale enhancer trap (ET screen and generated 338 insertions of the Tol2 transposon-based ET cassette into the zebrafish genome. These insertions were generated by remobilizing the transposon from two different donor sites in two transgenic lines. We found that 39% of Tol2 insertions occurred in transcription units, mostly into introns. Analysis of the transposon target sites revealed no strict specificity at the DNA sequence level. However, Tol2 was prone to target AT-rich regions with weak palindromic consensus sequences centered at the insertion site. Conclusion Our systematic analysis of sequential remobilizations of the Tol2 transposon from two independent sites within a vertebrate genome has revealed properties such as a tendency to integrate into transcription units and into AT-rich palindrome-like sequences. This information will influence the development of various applications involving DNA transposons and Tol2 in particular.

  3. Genome-wide analysis of Tol2 transposon reintegration in zebrafish.

    Science.gov (United States)

    Kondrychyn, Igor; Garcia-Lecea, Marta; Emelyanov, Alexander; Parinov, Sergey; Korzh, Vladimir

    2009-09-08

    Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome. We performed a large-scale enhancer trap (ET) screen and generated 338 insertions of the Tol2 transposon-based ET cassette into the zebrafish genome. These insertions were generated by remobilizing the transposon from two different donor sites in two transgenic lines. We found that 39% of Tol2 insertions occurred in transcription units, mostly into introns. Analysis of the transposon target sites revealed no strict specificity at the DNA sequence level. However, Tol2 was prone to target AT-rich regions with weak palindromic consensus sequences centered at the insertion site. Our systematic analysis of sequential remobilizations of the Tol2 transposon from two independent sites within a vertebrate genome has revealed properties such as a tendency to integrate into transcription units and into AT-rich palindrome-like sequences. This information will influence the development of various applications involving DNA transposons and Tol2 in particular.

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

    Science.gov (United States)

    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.

  5. A systematic identification of Kolobok superfamily transposons in Trichomonas vaginalis and sequence analysis on related transposases

    Institute of Scientific and Technical Information of China (English)

    Qingshu Meng; Kaifu Chen; Lina Ma; Songnian Hu; Jun Yu

    2011-01-01

    Transposons are sequence elements widely distributed among genomes of all three kingdoms of life, providing genomic changes and playing significant roles in genome evolution. Trichomonas vaginalis is an excellent model system for transposon study since its genome ( ~ 160 Mb) has been sequenced and is composed of ~65% transposons and other repetitive elements. In this study, we primarily report the identification of Kolobok-type transposons (termed tvBac) in T. vaginalis and the results of transposase sequence analysis. We categorized 24 novel subfamilies of the Kolobok element, including one autonomous subfamily and 23 non-autonomous subfamilies. We also identified a novel H2CH motif in tvBac transposases based on multiple sequence alignment. In addition, we supposed that tvBac and Mutator transposons may have evolved independently from a common ancestor according to our phylogenetic analysis. Our results provide basic information for the understanding of the function and evolution of tvBac transposons in particular and other related transposon families in general.

  6. Gene Therapy with the Sleeping Beauty Transposon System.

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    Kebriaei, Partow; Izsvák, Zsuzsanna; Narayanavari, Suneel A; Singh, Harjeet; Ivics, Zoltán

    2017-11-01

    The widespread clinical implementation of gene therapy requires the ability to stably integrate genetic information through gene transfer vectors in a safe, effective, and economical manner. The latest generation of Sleeping Beauty (SB) transposon vectors fulfills these requirements, and may overcome limitations associated with viral gene transfer vectors and transient nonviral gene delivery approaches that are prevalent in ongoing clinical trials. The SB system enables high-level stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, thereby representing a highly attractive gene transfer strategy for clinical use. Here, we review the most important aspects of using SB for gene therapy, including vectorization as well as genomic integration features. We also illustrate the path to successful clinical implementation by highlighting the application of chimeric antigen receptor (CAR)-modified T cells in cancer immunotherapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Suicidal autointegration of sleeping beauty and piggyBac transposons in eukaryotic cells.

    Directory of Open Access Journals (Sweden)

    Yongming Wang

    2014-03-01

    Full Text Available Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. 'Cut and paste' DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB and piggyBac (PB that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1, a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB.

  8. Evaluating Risks of Insertional Mutagenesis by DNA Transposons in Gene Therapy

    Science.gov (United States)

    Hackett, Perry B.; Largaespada, David A.; Switzer, Kirsten C.; Cooper, Laurence J.N.

    2013-01-01

    Investigational therapy can be successfully undertaken using viral- and non-viral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR+ T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease. PMID:23313630

  9. [Active miniature inverted-repeat transposable elements transposon in plants: a review].

    Science.gov (United States)

    Hu, Bingjie; Zhou, Mingbing

    2018-02-25

    Miniature inverted-repeat transposable elements transposon is a special transposon that could transpose by "cut-paste" mechanism, which is one of characteristics of DNA transposons. Otherwise, the copy number of MITEs is very high, which is one of characteristics of RNA transposons. Many MITE families have been reported, but little about active MITEs. We summarize recent advances in studying active MITEs. Most the MITEs belong to the Tourist-like family, such as mPing, mGing, PhTourist1, Tmi1 and PhTst-3. Additionally, DTstu1 and MITE-39 belong to Stowaway-like family, and AhMITEs1 belongs to Mutator-like family. Moreover, we summarize the structure (terminal inverse repeats and target site duplications), copy number, evolution pattern and transposition characteristics of these active MITEs, to provide the foundation for the identification of other active MITEs and subsequent research on MITE transposition and amplification mechanism.

  10. Stochastic Predator-Prey Dynamics of Transposons in the Human Genome

    Science.gov (United States)

    Xue, Chi; Goldenfeld, Nigel

    2016-11-01

    Transposable elements, or transposons, are DNA sequences that can jump from site to site in the genome during the life cycle of a cell, usually encoding the very enzymes which perform their excision. However, some transposons are parasitic, relying on the enzymes produced by the regular transposons. In this case, we show that a stochastic model, which takes into account the small copy numbers of the active transposons in a cell, predicts noise-induced predator-prey oscillations with a characteristic time scale that is much longer than the cell replication time, indicating that the state of the predator-prey oscillator is stored in the genome and transmitted to successive generations. Our work demonstrates the important role of the number fluctuations in the expression of mobile genetic elements, and shows explicitly how ecological concepts can be applied to the dynamics and fluctuations of living genomes.

  11. A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp. michiganensis.

    Science.gov (United States)

    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.

  12. Identifying transposon insertions and their effects from RNA-sequencing data.

    Science.gov (United States)

    de Ruiter, Julian R; Kas, Sjors M; Schut, Eva; Adams, David J; Koudijs, Marco J; Wessels, Lodewyk F A; Jonkers, Jos

    2017-07-07

    Insertional mutagenesis using engineered transposons is a potent forward genetic screening technique used to identify cancer genes in mouse model systems. In the analysis of these screens, transposon insertion sites are typically identified by targeted DNA-sequencing and subsequently assigned to predicted target genes using heuristics. As such, these approaches provide no direct evidence that insertions actually affect their predicted targets or how transcripts of these genes are affected. To address this, we developed IM-Fusion, an approach that identifies insertion sites from gene-transposon fusions in standard single- and paired-end RNA-sequencing data. We demonstrate IM-Fusion on two separate transposon screens of 123 mammary tumors and 20 B-cell acute lymphoblastic leukemias, respectively. We show that IM-Fusion accurately identifies transposon insertions and their true target genes. Furthermore, by combining the identified insertion sites with expression quantification, we show that we can determine the effect of a transposon insertion on its target gene(s) and prioritize insertions that have a significant effect on expression. We expect that IM-Fusion will significantly enhance the accuracy of cancer gene discovery in forward genetic screens and provide initial insight into the biological effects of insertions on candidate cancer genes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Transposon-mediated BAC transgenesis in human ES cells

    Science.gov (United States)

    Rostovskaya, Maria; Fu, Jun; Obst, Mandy; Baer, Isabell; Weidlich, Stefanie; Wang, Hailong; Smith, Andrew J. H.; Anastassiadis, Konstantinos; Stewart, A. Francis

    2012-01-01

    Transgenesis is a cornerstone of molecular biology. The ability to integrate a specifically engineered piece of DNA into the genome of a living system is fundamental to our efforts to understand life and exploit its implications for medicine, nanotechnology and bioprospecting. However, transgenesis has been hampered by position effects and multi-copy integration problems, which are mainly due to the use of small, plasmid-based transgenes. Large transgenes based on native genomic regions cloned into bacterial artificial chromosomes (BACs) circumvent these problems but are prone to fragmentation. Herein, we report that contrary to widely held notions, large BAC-sized constructs do not prohibit transposition. We also report the first reliable method for BAC transgenesis in human embryonic stem cells (hESCs). The PiggyBac or Sleeping Beauty transposon inverted repeats were integrated into BAC vectors by recombineering, followed by co-lipofection with the corresponding transposase in hESCs to generate robust fluorescent protein reporter lines for OCT4, NANOG, GATA4 and PAX6. BAC transposition delivers several advantages, including increased frequencies of single-copy, full-length integration, which will be useful in all transgenic systems but especially in difficult venues like hESCs. PMID:22753106

  14. Cut-and-Paste Transposons in Fungi with Diverse Lifestyles.

    Science.gov (United States)

    Muszewska, Anna; Steczkiewicz, Kamil; Stepniewska-Dziubinska, Marta; Ginalski, Krzysztof

    2017-12-01

    Transposable elements (TEs) shape genomes via recombination and transposition, lead to chromosomal rearrangements, create new gene neighborhoods, and alter gene expression. They play key roles in adaptation either to symbiosis in Amanita genus or to pathogenicity in Pyrenophora tritici-repentis. Despite growing evidence of their importance, the abundance and distribution of mobile elements replicating in a "cut-and-paste" fashion is barely described so far. In order to improve our knowledge on this old and ubiquitous class of transposable elements, 1,730 fungal genomes were scanned using both de novo and homology-based approaches. DNA TEs have been identified across the whole data set and display uneven distribution from both DNA TE classification and fungal taxonomy perspectives. DNA TE content correlates with genome size, which confirms that many transposon families proliferate simultaneously. In contrast, it is independent from intron density, average gene distance and GC content. TE count is associated with species' lifestyle and tends to be elevated in plant symbionts and decreased in animal parasites. Lastly, we found that fungi with both RIP and RNAi systems have more total DNA TE sequences but less elements retaining a functional transposase, what reflects stringent control over transposition. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  15. Horizontal transfers of Mariner transposons between mammals and insects

    Directory of Open Access Journals (Sweden)

    Oliveira Sarah G

    2012-09-01

    Full Text Available Abstract Background Active transposable elements (TEs can be passed between genomes of different species by horizontal transfer (HT. This may help them to avoid vertical extinction due to elimination by natural selection or silencing. HT is relatively frequent within eukaryotic taxa, but rare between distant species. Findings Closely related Mariner-type DNA transposon families, collectively named as Mariner-1_Tbel families, are present in the genomes of two ants and two mammalian genomes. Consensus sequences of the four families show pairwise identities greater than 95%. In addition, mammalian Mariner1_BT family shows a close evolutionary relationship with some insect Mariner families. Mammalian Mariner1_BT type sequences are present only in species from three groups including ruminants, tooth whales (Odontoceti, and New World leaf-nosed bats (Phyllostomidae. Conclusions Horizontal transfer accounts for the presence of Mariner_Tbel and Mariner1_BT families in mammals. Mariner_Tbel family was introduced into hedgehog and tree shrew genomes approximately 100 to 69 million years ago (MYA. Most likely, these TE families were transferred from insects to mammals, but details of the transfer remain unknown.

  16. Horizontal transfers of Mariner transposons between mammals and insects.

    Science.gov (United States)

    Oliveira, Sarah G; Bao, Weidong; Martins, Cesar; Jurka, Jerzy

    2012-09-26

    Active transposable elements (TEs) can be passed between genomes of different species by horizontal transfer (HT). This may help them to avoid vertical extinction due to elimination by natural selection or silencing. HT is relatively frequent within eukaryotic taxa, but rare between distant species. Closely related Mariner-type DNA transposon families, collectively named as Mariner-1_Tbel families, are present in the genomes of two ants and two mammalian genomes. Consensus sequences of the four families show pairwise identities greater than 95%. In addition, mammalian Mariner1_BT family shows a close evolutionary relationship with some insect Mariner families. Mammalian Mariner1_BT type sequences are present only in species from three groups including ruminants, tooth whales (Odontoceti), and New World leaf-nosed bats (Phyllostomidae). Horizontal transfer accounts for the presence of Mariner_Tbel and Mariner1_BT families in mammals. Mariner_Tbel family was introduced into hedgehog and tree shrew genomes approximately 100 to 69 million years ago (MYA). Most likely, these TE families were transferred from insects to mammals, but details of the transfer remain unknown.

  17. Radiation and transposon-induced genetic damage in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Balter, H.; Griffith, C.S.; American Museum of Natural History, New York; Margulies, L.

    1992-01-01

    The interaction of X-ray-induced and transposon-induced damage was investigated in P-M hybrid dysgenesis in Drosophila melanogaster. X-ray dose-response of 330-1320 rad was monitored for sterility, fecundicity and partial X/Y chromosome loss among F 2 progeny derived from dysgenic cross of M strain females xP strain males (cross A) and its reciprocal (cross B), using a weaker and the standard Harwich P strain subline. The synergistic effect of P element activity and X-rays on sterility was observed only in cross A hybrids and the dose-response was nonlinear in hybrids derived from the strong standard reference Harwich subline, H W . This finding suggests that lesions induced by both mutator systems which produce the synergistic effects are 2-break events. Effect of increasing dose on the decline of fecundicity was synergistic, but linear, in hybrids of either subline. There was no interaction evident and thus no synergism in X/Y nondisjunction and partial Y chromosome loss measured by the loss of the B s marker alone or together with the y + marker. Interaction was detected in the loss of the y + marker alone from the X and Y chromosomes. The possible three-way interaction of X-rays (660 rad), post-replication repair deficiency and P elements mobility was assessed by measuring transmission distortion in dysgenic males derived from the Π 2 P strain. (author). 38 refs.; 5 tabs

  18. Construction of a large-scale Burkholderia cenocepacia J2315 transposon mutant library

    Science.gov (United States)

    Wong, Yee-Chin; Pain, Arnab; Nathan, Sheila

    2014-09-01

    Burkholderia cenocepacia, a pathogenic member of the Burkholderia cepacia complex (Bcc), has emerged as a significant threat towards cystic fibrosis patients, where infection often leads to the fatal clinical manifestation known as cepacia syndrome. Many studies have investigated the pathogenicity of B. cenocepacia as well as its ability to become highly resistant towards many of the antibiotics currently in use. In addition, studies have also been undertaken to understand the pathogen's capacity to adapt and survive in a broad range of environments. Transposon based mutagenesis has been widely used in creating insertional knock-out mutants and coupled with recent advances in sequencing technology, robust tools to study gene function in a genome-wide manner have been developed based on the assembly of saturated transposon mutant libraries. In this study, we describe the construction of a large-scale library of B. cenocepacia transposon mutants. To create transposon mutants of B. cenocepacia strain J2315, electrocompetent bacteria were electrotransformed with the EZ-Tn5 transposome. Tetracyline resistant colonies were harvested off selective agar and pooled. Mutants were generated in multiple batches with each batch consisting of ˜20,000 to 40,000 mutants. Transposon insertion was validated by PCR amplification of the transposon region. In conclusion, a saturated B. cenocepacia J2315 transposon mutant library with an estimated total number of 500,000 mutants was successfully constructed. This mutant library can now be further exploited as a genetic tool to assess the function of every gene in the genome, facilitating the discovery of genes important for bacterial survival and adaptation, as well as virulence.

  19. Novel Aminoglycoside Resistance Transposons and Transposon-Derived Circular Forms Detected in Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates

    Science.gov (United States)

    Dwibedi, Chinmay Kumar; Sjöström, Karin; Edquist, Petra; Wai, Sun Nyunt; Uhlin, Bernt Eric

    2016-01-01

    Acinetobacter baumannii has emerged as an important opportunistic pathogen equipped with a growing number of antibiotic resistance genes. Our study investigated the molecular epidemiology and antibiotic resistance features of 28 consecutive carbapenem-resistant clinical isolates of A. baumannii collected throughout Sweden in 2012 and 2013. The isolates mainly belonged to clonal complexes (CCs) with an extensive international distribution, such as CC2 (n = 16) and CC25 (n = 7). Resistance to carbapenems was related to blaOXA-23 (20 isolates), blaOXA-24/40-like (6 isolates), blaOXA-467 (1 isolate), and ISAba1-blaOXA-69 (1 isolate). Ceftazidime resistance was associated with blaPER-7 in the CC25 isolates. Two classical point mutations were responsible for resistance to quinolones in all the isolates. Isolates with high levels of resistance to aminoglycosides carried the 16S rRNA methylase armA gene. The isolates also carried a variety of genes encoding aminoglycoside-modifying enzymes. Several novel structures involved in aminoglycoside resistance were identified, including Tn6279, ΔTn6279, Ab-ST3-aadB, and different assemblies of Tn6020 and TnaphA6. Importantly, a number of circular forms related to the IS26 or ISAba125 composite transposons were detected. The frequent occurrence of these circular forms in the populations of several isolates indicates a potential role of these circular forms in the dissemination of antibiotic resistance genes. PMID:26824943

  20. Bursts and horizontal evolution of DNA transposons in the speciation of pseudotetraploid salmonids

    Directory of Open Access Journals (Sweden)

    Davidson William S

    2007-11-01

    Full Text Available Abstract Background Several genome duplications have occurred in the evolutionary history of teleost fish. In returning to a stable diploid state, the polyploid genome reorganized, and large portions are lost, while the fish lines evolved to numerous species. Large scale transposon movement has been postulated to play an important role in the genome reorganization process. We analyzed the DNA sequence of several large loci in Salmo salar and other species for the presence of DNA transposon families. Results We have identified bursts of activity of 14 families of DNA transposons (12 Tc1-like and 2 piggyBac-like families, including 11 novel ones in genome sequences of Salmo salar. Several of these families have similar sequences in a number of closely and distantly related fish, lamprey, and frog species as well as in the parasite Schistosoma japonicum. Analysis of sequence similarities between copies within the families of these bursts demonstrates several waves of transposition activities coinciding with salmonid species divergence. Tc1-like families show a master gene-like copying process, illustrated by extensive but short burst of copying activity, while the piggyBac-like families show a more random copying pattern. Recent families may include copies with an open reading frame for an active transposase enzyme. Conclusion We have identified defined bursts of transposon activity that make use of master-slave and random mechanisms. The bursts occur well after hypothesized polyploidy events and coincide with speciation events. Parasite-mediated lateral transfer of transposons are implicated.

  1. Genome-scale metabolic network validation of Shewanella oneidensis using transposon insertion frequency analysis.

    Directory of Open Access Journals (Sweden)

    Hong Yang

    2014-09-01

    Full Text Available Transposon mutagenesis, in combination with parallel sequencing, is becoming a powerful tool for en-masse mutant analysis. A probability generating function was used to explain observed miniHimar transposon insertion patterns, and gene essentiality calls were made by transposon insertion frequency analysis (TIFA. TIFA incorporated the observed genome and sequence motif bias of the miniHimar transposon. The gene essentiality calls were compared to: 1 previous genome-wide direct gene-essentiality assignments; and, 2 flux balance analysis (FBA predictions from an existing genome-scale metabolic model of Shewanella oneidensis MR-1. A three-way comparison between FBA, TIFA, and the direct essentiality calls was made to validate the TIFA approach. The refinement in the interpretation of observed transposon insertions demonstrated that genes without insertions are not necessarily essential, and that genes that contain insertions are not always nonessential. The TIFA calls were in reasonable agreement with direct essentiality calls for S. oneidensis, but agreed more closely with E. coli essentiality calls for orthologs. The TIFA gene essentiality calls were in good agreement with the MR-1 FBA essentiality predictions, and the agreement between TIFA and FBA predictions was substantially better than between the FBA and the direct gene essentiality predictions.

  2. The expanding universe of transposon technologies for gene and cell engineering.

    Science.gov (United States)

    Ivics, Zoltán; Izsvák, Zsuzsanna

    2010-12-07

    Transposable elements can be viewed as natural DNA transfer vehicles that, similar to integrating viruses, are capable of efficient genomic insertion. The mobility of class II transposable elements (DNA transposons) can be controlled by conditionally providing the transposase component of the transposition reaction. Thus, a DNA of interest (be it a fluorescent marker, a small hairpin (sh)RNA expression cassette, a mutagenic gene trap or a therapeutic gene construct) cloned between the inverted repeat sequences of a transposon-based vector can be used for stable genomic insertion in a regulated and highly efficient manner. This methodological paradigm opened up a number of avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture, the production of germline transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species, and therapy of genetic disorders in humans. Sleeping Beauty (SB) was the first transposon shown to be capable of gene transfer in vertebrate cells, and recent results confirm that SB supports a full spectrum of genetic engineering including transgenesis, insertional mutagenesis, and therapeutic somatic gene transfer both ex vivo and in vivo. The first clinical application of the SB system will help to validate both the safety and efficacy of this approach. In this review, we describe the major transposon systems currently available (with special emphasis on SB), discuss the various parameters and considerations pertinent to their experimental use, and highlight the state of the art in transposon technology in diverse genetic applications.

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

    Directory of Open Access Journals (Sweden)

    Rita eMonson

    2015-12-01

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

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

    Science.gov (United States)

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

    2016-07-01

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

  5. Transcription and somatic transposition of the maize En/Spm transposon system in rice

    NARCIS (Netherlands)

    Greco, R.; Ouwerkerk, P.B.F.; Taal, A.J.C.; Sallaud, C.; Guiderdoni, E.; Meijer, A.H.; Hoge, J.H.C.; Pereira, A.B.

    2004-01-01

    Transposition of the maize En/Spm system in rice was investigated using a two-component construct consisting of an immobilised transposase source driven by the CaMV 35S-promoter, and a modified I/dSpm transposon. Mobilization of I/dSpm in somatic sectors was demonstrated by sequencing of excision

  6. Mos1 transposon-based transformation of fish cell lines using baculoviral vectors

    International Nuclear Information System (INIS)

    Yokoo, Masako; Fujita, Ryosuke; Nakajima, Yumiko; Yoshimizu, Mamoru; Kasai, Hisae; Asano, Shin-ichiro; Bando, Hisanori

    2013-01-01

    Highlights: •The baculovirus vector infiltrates the cells of economic important fishes. •Drosophila Mos1 transposase expressed in fish cells maintains its ability to localize to the nucleus. •The baculoviral vector carrying Mos1 is a useful tool to stably transform fish cells. -- Abstract: Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells

  7. Mos1 transposon-based transformation of fish cell lines using baculoviral vectors

    Energy Technology Data Exchange (ETDEWEB)

    Yokoo, Masako [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Fujita, Ryosuke [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Innate Immunity Laboratory, Graduate School of Life Science and Creative Research Institution, Hokkaido University, Sapporo 001-0021 (Japan); Nakajima, Yumiko [Functional Genomics Group, COMB, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa 903-0213 (Japan); Yoshimizu, Mamoru; Kasai, Hisae [Faculty of Fisheries Sciences, Hokkaido University, Hakodate 041-8611 (Japan); Asano, Shin-ichiro [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan); Bando, Hisanori, E-mail: hban@abs.agr.hokudai.ac.jp [Laboratory of Applied Molecular Entomology, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589 (Japan)

    2013-09-13

    Highlights: •The baculovirus vector infiltrates the cells of economic important fishes. •Drosophila Mos1 transposase expressed in fish cells maintains its ability to localize to the nucleus. •The baculoviral vector carrying Mos1 is a useful tool to stably transform fish cells. -- Abstract: Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.

  8. Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH

    Directory of Open Access Journals (Sweden)

    Burgis Timothy A

    2009-07-01

    Full Text Available Abstract Background In recent years there has been an increasing problem with Staphylococcus aureus strains that are resistant to treatment with existing antibiotics. An important starting point for the development of new antimicrobial drugs is the identification of "essential" genes that are important for bacterial survival and growth. Results We have developed a robust microarray and PCR-based method, Transposon-Mediated Differential Hybridisation (TMDH, that uses novel bioinformatics to identify transposon inserts in genome-wide libraries. Following a microarray-based screen, genes lacking transposon inserts are re-tested using a PCR and sequencing-based approach. We carried out a TMDH analysis of the S. aureus genome using a large random mariner transposon library of around a million mutants, and identified a total of 351 S. aureus genes important for survival and growth in culture. A comparison with the essential gene list experimentally derived for Bacillus subtilis highlighted interesting differences in both pathways and individual genes. Conclusion We have determined the first comprehensive list of S. aureus essential genes. This should act as a useful starting point for the identification of potential targets for novel antimicrobial compounds. The TMDH methodology we have developed is generic and could be applied to identify essential genes in other bacterial pathogens.

  9. Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations.

    Science.gov (United States)

    Maglennon, Gareth A; Cook, Beth S; Deeney, Alannah S; Bossé, Janine T; Peters, Sarah E; Langford, Paul R; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N

    2013-12-21

    Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.

  10. A putative autonomous 20.5 kb-CACTA transposon insertion in an F3'H allele identifies a new CACTA transposon subfamily in Glycine max

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    Vodkin Lila

    2008-12-01

    Full Text Available Abstract Background The molecular organization of very few genetically defined CACTA transposon systems have been characterized thoroughly as those of Spm/En in maize, Tam1 of Antirrhinum majus Candystripe1 (Cs1 from Sorghum bicolor and CAC1 from Arabidopsis thaliana, for example. To date, only defective deletion derivatives of CACTA elements have been described for soybean, an economically important plant species whose genome sequence will be completed in 2008. Results We identified a 20.5 kb insertion in a soybean flavonoid 3'-hydroxylase (F3'H gene representing the t* allele (stable gray trichome color whose origin traces to a single mutable chimeric plant displaying both tawny and gray trichomes. This 20.5 kb insertion has the molecular structure of a putative autonomous transposon of the CACTA family, designated Tgmt*. It encodes a large gene that was expressed in two sister isolines (T* and tm of the stable gray line (t* from which Tgmt* was isolated. RT-PCR derived cDNAs uncovered the structure of a large precursor mRNA as well as alternatively spliced transcripts reminiscent of the TNPA-mRNA generated by the En-1 element of maize but without sequence similarity to the maize TNPA. The larger mRNA encodes a transposase with a tnp2 and TNP1-transposase family domains. Because the two soybean lines expressing Tgmt* were derived from the same mutable chimeric plant that created the stable gray trichome t* allele line from which the element was isolated, Tgmt* has the potential to be an autonomous element that was rapidly inactivated in the stable gray trichome t* line. Comparison of Tgmt* to previously described Tgm elements demonstrated that two subtypes of CACTA transposon families exist in soybean based on divergence of their characteristic subterminal repeated motifs and their transposases. In addition, we report the sequence and annotation of a BAC clone containing the F3'H gene (T locus which was interrupted by the novel Tgmt* element

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

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    Maura McGrail

    2011-04-01

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

  12. The Nucleoid Binding Protein H-NS Biases Genome-Wide Transposon Insertion Landscapes

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    Satoshi Kimura

    2016-08-01

    Full Text Available Transposon insertion sequencing (TIS; also known as TnSeq is a potent approach commonly used to comprehensively define the genetic loci that contribute to bacterial fitness in diverse environments. A key presumption underlying analyses of TIS datasets is that loci with a low frequency of transposon insertions contribute to fitness. However, it is not known whether factors such as nucleoid binding proteins can alter the frequency of transposon insertion and thus whether TIS output may systematically reflect factors that are independent of the role of the loci in fitness. Here, we investigated whether the histone-like nucleoid structuring (H-NS protein, which preferentially associates with AT-rich sequences, modulates the frequency of Mariner transposon insertion in the Vibrio cholerae genome, using comparative analysis of TIS results from wild-type (wt and Δhns V. cholerae strains. These analyses were overlaid on gene classification based on GC content as well as on extant genome-wide identification of H-NS binding loci. Our analyses revealed a significant dearth of insertions within AT-rich loci in wt V. cholerae that was not apparent in the Δhns insertion library. Additionally, we observed a striking correlation between genetic loci that are overrepresented in the Δhns insertion library relative to their insertion frequency in wt V. cholerae and loci previously found to physically interact with H-NS. Collectively, our findings reveal that factors other than genetic fitness can systematically modulate the frequency of transposon insertions in TIS studies and add a cautionary note to interpretation of TIS data, particularly for AT-rich sequences.

  13. The expanding universe of transposon technologies for gene and cell engineering

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    Ivics Zoltán

    2010-12-01

    Full Text Available Abstract Transposable elements can be viewed as natural DNA transfer vehicles that, similar to integrating viruses, are capable of efficient genomic insertion. The mobility of class II transposable elements (DNA transposons can be controlled by conditionally providing the transposase component of the transposition reaction. Thus, a DNA of interest (be it a fluorescent marker, a small hairpin (shRNA expression cassette, a mutagenic gene trap or a therapeutic gene construct cloned between the inverted repeat sequences of a transposon-based vector can be used for stable genomic insertion in a regulated and highly efficient manner. This methodological paradigm opened up a number of avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture, the production of germline transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species, and therapy of genetic disorders in humans. Sleeping Beauty (SB was the first transposon shown to be capable of gene transfer in vertebrate cells, and recent results confirm that SB supports a full spectrum of genetic engineering including transgenesis, insertional mutagenesis, and therapeutic somatic gene transfer both ex vivo and in vivo. The first clinical application of the SB system will help to validate both the safety and efficacy of this approach. In this review, we describe the major transposon systems currently available (with special emphasis on SB, discuss the various parameters and considerations pertinent to their experimental use, and highlight the state of the art in transposon technology in diverse genetic applications.

  14. Mariner transposons are sailing in the genome of the blood-sucking bug Rhodnius prolixus.

    Science.gov (United States)

    Filée, Jonathan; Rouault, Jacques-Deric; Harry, Myriam; Hua-Van, Aurélie

    2015-12-15

    The Triatomine bug Rhodnius prolixus is a vector of Trypanosoma cruzi, which causes the Chagas disease in Latin America. R. prolixus can also transfer transposable elements horizontally across a wide range of species. We have taken advantage of the availability of the 700 Mbp complete genome sequence of R. prolixus to study the dynamics of invasion and persistence of transposable elements in this species. Using both library-based and de novo methods of transposon detection, we found less than 6 % of transposable elements in the R. prolixus genome, a relatively low percentage compared to other insect genomes with a similar genome size. DNA transposons are surprisingly abundant and elements belonging to the mariner family are by far the most preponderant components of the mobile part of this genome with 11,015 mariner transposons that could be clustered in 89 groups (75 % of the mobilome). Our analysis allowed the detection of a new mariner clade in the R. prolixus genome, that we called nosferatis. We demonstrated that a large diversity of mariner elements invaded the genome and expanded successfully over time via three main processes. (i) several families experienced recent and massive expansion, for example an explosive burst of a single mariner family led to the generation of more than 8000 copies. These recent expansion events explain the unusual prevalence of mariner transposons in the R. prolixus genome. Other families expanded via older bursts of transposition demonstrating the long lasting permissibility of mariner transposons in the R. prolixus genome. (ii) Many non-autonomous families generated by internal deletions were also identified. Interestingly, two non autonomous families were generated by atypical recombinations (5' part replacement with 3' part). (iii) at least 10 cases of horizontal transfers were found, supporting the idea that host/vector relationships played a pivotal role in the transmission and subsequent persistence of transposable

  15. Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum.

    Science.gov (United States)

    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

  16. Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells

    DEFF Research Database (Denmark)

    Kues, Wilfried A.; Herrmann, Doris; Barg-Kues, Brigitte

    2013-01-01

    the nonviral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4, and cMyc. Successful reprogramming to a pluripotent state was indicated by changes in cell morphology and reactivation of the Oct4-EGFP reporter. The transposon-reprogrammed induced pluripotent stem (i......PS) cells showed long-term proliferation in vitro over >40 passages, expressed transcription factors typical of embryonic stem cells, including OCT4, NANOG, SOX2, REX1, ESRRB, DPPA5, and UTF1 and surface markers of pluripotency, including SSEA-1 and TRA-1-60. In vitro differentiation resulted in derivatives......The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing...

  17. Hybrid Lentivirus-transposon Vectors With a Random Integration Profile in Human Cells

    DEFF Research Database (Denmark)

    Staunstrup, Nicklas H; Moldt, Brian; Mátés, Lajos

    2009-01-01

    Gene delivery by human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (LVs) is efficient, but genomic integration of the viral DNA is strongly biased toward transcriptionally active loci resulting in an increased risk of insertional mutagenesis in gene therapy protocols. Nonviral...... Sleeping Beauty (SB) transposon vectors have a significantly safer insertion profile, but efficient delivery into relevant cell/tissue types is a limitation. In an attempt to combine the favorable features of the two vector systems we established a novel hybrid vector technology based on SB transposase......-mediated insertion of lentiviral DNA circles generated during transduction of target cells with integrase (IN)-defective LVs (IDLVs). By construction of a lentivirus-transposon hybrid vector allowing transposition exclusively from circular viral DNA substrates, we demonstrate that SB transposase added in trans...

  18. The rde-1 gene, RNA interference, and transposon silencing in C. elegans.

    Science.gov (United States)

    Tabara, H; Sarkissian, M; Kelly, W G; Fleenor, J; Grishok, A; Timmons, L; Fire, A; Mello, C C

    1999-10-15

    Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.

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

    Science.gov (United States)

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

    2015-01-01

    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

  20. Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum.

    OpenAIRE

    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 tra...

  1. TCUP: A novel hAT transposon active in maize tissue culture

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    Alan eSmith

    2012-01-01

    Full Text Available Transposable elements are capable of inducing heritable de novo genetic variation. The sequences capable of reactivation, and environmental factors that induce mobilization, remain poorly defined even in well-studied genomes such as maize. We treated maize tissue culture with the demethylating agent 5-aza-2-deoxcytidine and examined long-term tissue culture lines to discover silenced transposable elements that have the potential to induce heritable genetic variation. Through these screens we have identified a novel low copy number hAT transposon, Tissue Culture Up-Regulated (TCUP, which is transcribed at high levels in long-term maize Black Mexican Sweet (BMS tissue culture and up-regulated in response to treatment with 5-aza-2-deoxycytidine. Analysis of the TIGR Maize Gene Index revealed that this element is the most frequently represented EST from the BMS cell culture library and is not represented in other tissue libraries, which is the basis for its name. A full-length sequence was assembled in inbred B73 that contains the putative functional motifs required for autonomous movement of a hAT transposon. Transposon display detected movement of TCUP in two long-term tissue cultured cell lines of the genotype Hi-II AxB and BMS. This research implicates TCUP as a transposon that is capable of reactivation and which may also be particularly sensitive to the stress of the tissue culture environment. Our findings are consistent with the hypothesis that epigenetic alterations potentiate genomic responses to stress during clonal propagation of plants.

  2. Sequence-indexed mutations in maize using the UniformMu transposon-tagging population

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    Baier John

    2007-05-01

    Full Text Available Abstract Background Gene knockouts are a critical resource for functional genomics. In Arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic DNA flanking insertion mutants. These Flanking Sequence Tags (FSTs map each mutant to a specific locus within the genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also generate somatic insertions that fail to segregate in subsequent generations. Results Transposon insertion sites from 106 UniformMu FSTs were tested for inheritance by locus-specific PCR. We confirmed 89% of the FSTs to be germinal transposon insertions. We found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. Instead, this subset of insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic sequences. The locus-specific PCR assays identified a knockout of a 6-phosphogluconate dehydrogenase gene that co-segregates with a seed mutant phenotype. The mutant phenotype linked to this knockout generates novel hypotheses about the role for the plastid-localized oxidative pentose phosphate pathway during grain-fill. Conclusion We show that FSTs from the UniformMu population identify stable, germinal insertion sites in maize. Moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis. We conclude from these data that the current collection of 1,882 non-redundant insertion sites from UniformMu provide a genome-wide resource for reverse genetics.

  3. The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

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    Boris Troyanovsky

    2016-01-01

    Full Text Available Minimal piggyBac vectors are a modified single-plasmid version of the classical piggyBac delivery system that can be used for stable transgene integration. These vectors have a truncated terminal domain in the delivery cassette and thus, integrate significantly less flanking transposon DNA into host cell chromatin than classical piggyBac vectors. Herein, we test various characteristics of this modified transposon. The integration efficiency of minimal piggyBac vectors was inversely related to the size of both the transposon and the entire plasmid, but inserts as large as 15 kb were efficiently integrated. Open and super-coiled vectors demonstrated the same integration efficiency while DNA methylation decreased the integration efficiency and silenced the expression of previously integrated sequences in some cell types. Importantly, the incidence of plasmid backbone integration was not increased above that seen in nontransposon control vectors. In BALB/c mice, we demonstrated prolonged expression of two transgenes (intracellular mCherry and secretable Gaussia luciferase when delivered by the minimal piggyBac that resulted in a more sustained antibody production against the immunogenic luciferase than when delivered by a transient (nontransposon vector plasmid. We conclude that minimal piggyBac vectors are an effective alternative to other integrative systems for stable DNA delivery in vitro and in vivo.

  4. Induction of rat liver tumor using the Sleeping Beauty transposon and electroporation.

    Science.gov (United States)

    Park, June-Shine; Kim, Bae-Hwan; Park, Sung Goo; Jung, Sun Young; Lee, Do Hee; Son, Woo-Chan

    2013-05-10

    The Sleeping Beauty (SB) transposon system has been receiving much attention as a gene transfer method of choice since it allows permanent gene expression after insertion into the host chromosome. However, low transposition frequency in higher eukaryotes limits its use in commonly-used mammalian species. Researchers have therefore attempted to modify gene delivery and expression to overcome this limitation. In mouse liver, tumor induction using SB introduced by the hydrodynamic method has been successfully accomplished. Liver tumor in rat models using SB could also be of great use; however, dose of DNA, injection volume, rate of injection and achieving back pressure limit the use of the hydrodynamics-based gene delivery. In the present study, we combined the electroporation, a relatively simple and easy gene delivery method, with the SB transposon system and as a result successfully induced tumor in rat liver by directly injecting the c-Myc, HRAS and shp53 genes. The tumor phenotype was determined as a sarcomatoid carcinoma. To our knowledge, this is the first demonstration of induction of tumor in the rat liver using the electroporation-enhanced SB transposon system. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Induction of transposon TN1 translocation under the action of different mutagens

    International Nuclear Information System (INIS)

    Kubanejshvili, M.G.; Smirnov, S.P.; Tarasov, V.A.

    1983-01-01

    Migration of ampicillin transposon Tn1 under normal conditions in Escherichia coli cells proceeds with low frequency (10 -4 transpositions for cell). The low transposition frequency is conditioned by the transposition repression, realized by the gene-repressor in transposon structure and, probably, by other regulating genes of the bacterium-host. E. coli cell treatment by physical and chemical mutagens resulted in induction of translocation of ampicillin transposon Tn1 from plasmid RP4 into other replicons. Mitomycin C and ultraviolet radiation produced stronger inducing effect as compared to nitroso-guanidine (NG). The effect of the given mutagens on transposition Tn1 correlated with their activating capacity with respect to inducible SOS-functions of E coli. The mutation of rec A didn't influence on spontaneous Tn1 transposition, but blocked completely the induction of transposition process under mutagen effect. The relationship of inducible transposition with SOS-functions in E. coli cells, controlled by recA and lexA genes, as well as the possible role of the process in genetic microorganism variability are discussed in the paper

  6. Chemical mutagens, transposons, and transgenes to interrogate gene function in Drosophila melanogaster.

    Science.gov (United States)

    Venken, Koen J T; Bellen, Hugo J

    2014-06-15

    The study of genetics, genes, and chromosomal inheritance was initiated by Thomas Morgan in 1910, when the first visible mutations were identified in fruit flies. The field expanded upon the work initiated by Herman Muller in 1926 when he used X-rays to develop the first balancer chromosomes. Today, balancers are still invaluable to maintain mutations and transgenes but the arsenal of tools has expanded vastly and numerous new methods have been developed, many relying on the availability of the genome sequence and transposable elements. Forward genetic screens based on chemical mutagenesis or transposable elements have resulted in the unbiased identification of many novel players involved in processes probed by specific phenotypic assays. Reverse genetic approaches have relied on the availability of a carefully selected set of transposon insertions spread throughout the genome to allow the manipulation of the region in the vicinity of each insertion. Lastly, the ability to transform Drosophila with single copy transgenes using transposons or site-specific integration using the ΦC31 integrase has allowed numerous manipulations, including the ability to create and integrate genomic rescue constructs, generate duplications, RNAi knock-out technology, binary expression systems like the GAL4/UAS system as well as other methods. Here, we will discuss the most useful methodologies to interrogate the fruit fly genome in vivo focusing on chemical mutagenesis, transposons and transgenes. Genome engineering approaches based on nucleases and RNAi technology are discussed in following chapters. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. An Enterobacter plasmid as a new genetic background for the transposon Tn1331

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    Alavi MR

    2011-11-01

    Full Text Available Mohammad R Alavi1,2, Vlado Antonic2, Adrien Ravizee1, Peter J Weina3, Mina Izadjoo1,2, Alexander Stojadinovic21Division of Wound Biology and Translational Research, Armed Forces Institute of Pathology and American Registry of Pathology, Washington DC, 2Combat Wound Initiative Program, Walter Reed Army Medical Center, Washington DC, 3The Walter Reed Army Institute of Research, Silver Spring, MD, USABackground: Genus Enterobacter includes important opportunistic nosocomial pathogens that could infect complex wounds. The presence of antibiotic resistance genes in these microorganisms represents a challenging clinical problem in the treatment of these wounds. In the authors’ screening of antibiotic-resistant bacteria from complex wounds, an Enterobacter species was isolated that harbors antibiotic-resistant plasmids conferring resistance to Escherichia coli. The aim of this study was to identify the resistance genes carried by one of these plasmids.Methods: The plasmids from the Enterobacter isolate were propagated in E. coli and one of the plasmids, designated as pR23, was sequenced by the Sanger method using fluorescent dye-terminator chemistry on a genetic analyzer. The assembled sequence was annotated by search of the GenBank database.Results: Plasmid pR23 is composed of the transposon Tn1331 and a backbone plasmid that is identical to the plasmid pPIGDM1 from Enterobacter agglomerans. The multidrug-resistance transposon Tn1331, which confers resistance to aminoglycoside and beta lactam antibiotics, has been previously isolated only from Klebsiella. The Enterobacter plasmid pPIGDM1, which carries a ColE1-like origin of replication and has no apparent selective marker, appears to provide a backbone for propagation of Tn1331 in Enterobacter. The recognition sequence of Tn1331 transposase for insertion into pPIGDM1 is the pentanucleotide TATTA, which occurs only once throughout the length of this plasmid.Conclusion: Transposition of Tn1331 into

  8. The bandit, a new DNA transposon from a hookworm-possible horizontal genetic transfer between host and parasite.

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    Thewarach Laha

    2007-09-01

    Full Text Available An enhanced understanding of the hookworm genome and its resident mobile genetic elements should facilitate understanding of the genome evolution, genome organization, possibly host-parasite co-evolution and horizontal gene transfer, and from a practical perspective, development of transposon-based transgenesis for hookworms and other parasitic nematodes.A novel mariner-like element (MLE was characterized from the genome of the dog hookworm, Ancylostoma caninum, and termed bandit. The consensus sequence of the bandit transposon was 1,285 base pairs (bp in length. The new transposon was flanked by perfect terminal inverted repeats of 32 nucleotides in length with a common target site duplication TA, and it encoded an open reading frame (ORF of 342 deduced amino acid residues. Phylogenetic comparisons confirmed that the ORF encoded a mariner-like transposase, which included conserved catalytic domains, and that the bandit transposon belonged to the cecropia subfamily of MLEs. The phylogenetic analysis also indicated that the Hsmar1 transposon from humans was the closest known relative of bandit, and that bandit and Hsmar1 constituted a clade discrete from the Tc1 subfamily of MLEs from the nematode Caenorhabditis elegans. Moreover, homology models based on the crystal structure of Mos1 from Drosophila mauritiana revealed closer identity in active site residues of the catalytic domain including Ser281, Lys289 and Asp293 between bandit and Hsmar1 than between Mos1 and either bandit or Hsmar1. The entire bandit ORF was amplified from genomic DNA and a fragment of the bandit ORF was amplified from RNA, indicating that this transposon is actively transcribed in hookworms.A mariner-like transposon termed bandit has colonized the genome of the hookworm A. caninum. Although MLEs exhibit a broad host range, and are identified in other nematodes, the closest phylogenetic relative of bandit is the Hsmar1 element of humans. This surprising finding suggests

  9. Structural basis of hAT transposon end recognition by Hermes, an octameric DNA transposase from Musca domestica.

    Science.gov (United States)

    Hickman, Alison B; Ewis, Hosam E; Li, Xianghong; Knapp, Joshua A; Laver, Thomas; Doss, Anna-Louise; Tolun, Gökhan; Steven, Alasdair C; Grishaev, Alexander; Bax, Ad; Atkinson, Peter W; Craig, Nancy L; Dyda, Fred

    2014-07-17

    Hermes is a member of the hAT transposon superfamily that has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. Although isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple nonspecific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility.

    Science.gov (United States)

    Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K; Quach, Helen Ngoc Bao; Joshi, Adita; LaFave, Matthew C; Burgess, Shawn M; Sampath, Karuna

    2016-04-07

    DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering. Copyright © 2016 Vrljicak et al.

  11. Rim 2/Hipa CACTA transposon display ; A new genetic marker technique in Oryza species

    Directory of Open Access Journals (Sweden)

    Lee Ju

    2005-03-01

    Full Text Available Abstract Background Transposons constitute the major fractions of repetitive sequences in eukaryotes, and have been crucial in the shaping of current genomes. Transposons are generally divided into two classes according to the mechanism underlying their transposition: RNA intermediate class 1 and DNA intermediate class 2. CACTA is a class 2 transposon superfamily, which is found exclusively in plants. As some transposons, including the CACTA superfamily, are highly abundant in plant species, and their nucleotide sequences are highly conserved within a family, they can be utilized as genetic markers, using a slightly modified version of the conventional AFLP protocol. Rim2 /Hipa is a CACTA transposon family having 16 bp consensus TIR sequences to be present in high copy numbers in rice genome. This research was carried out in order to develop a Rim2/Hipa CACTA-AFLP or Rim2/Hipa CACTA-TD (transposon display, hereafter Rim2/Hipa-TD protocol for the study of genetic markers in map construction and the study of genetic diversity in rice. Results Rim2/Hipa-TD generated ample polymorphic profiles among the different rice accessions, and the amplification profiles were highly reproducible between different thermocyclers and Taq polymerases. These amplification profiles allowed for clear distinction between two different ecotypes, Japonica and Indica, of Oryza sativa. In the analysis of RIL populations, the Rim2/Hipa-TD markers were found to be segregated largely in a dominant manner, although in a few cases, non-parental bands were observed in the segregating populations. Upon linkage analysis, the Rim2/Hipa-TD markers were found to be distributed in the regions proximal to the centromeres of the chromosomes. The distribution of the Rim2/Hipa CACTA elements was surveyed in 15 different Oryza species via Rim2/Hipa-TD. While Rim2/Hipa-TD yielded ample amplification profiles between 100 to 700 bp in the AA diploid Oryza species, other species having BB, CC

  12. The transposon Galileo generates natural chromosomal inversions in Drosophila by ectopic recombination.

    Science.gov (United States)

    Delprat, Alejandra; Negre, Bàrbara; Puig, Marta; Ruiz, Alfredo

    2009-11-18

    Transposable elements (TEs) are responsible for the generation of chromosomal inversions in several groups of organisms. However, in Drosophila and other Dipterans, where inversions are abundant both as intraspecific polymorphisms and interspecific fixed differences, the evidence for a role of TEs is scarce. Previous work revealed that the transposon Galileo was involved in the generation of two polymorphic inversions of Drosophila buzzatii. To assess the impact of TEs in Drosophila chromosomal evolution and shed light on the mechanism involved, we isolated and sequenced the two breakpoints of another widespread polymorphic inversion from D. buzzatii, 2z(3). In the non inverted chromosome, the 2z(3) distal breakpoint was located between genes CG2046 and CG10326 whereas the proximal breakpoint lies between two novel genes that we have named Dlh and Mdp. In the inverted chromosome, the analysis of the breakpoint sequences revealed relatively large insertions (2,870-bp and 4,786-bp long) including two copies of the transposon Galileo (subfamily Newton), one at each breakpoint, plus several other TEs. The two Galileo copies: (i) are inserted in opposite orientation; (ii) present exchanged target site duplications; and (iii) are both chimeric. Our observations provide the best evidence gathered so far for the role of TEs in the generation of Drosophila inversions. In addition, they show unequivocally that ectopic recombination is the causative mechanism. The fact that the three polymorphic D. buzzatii inversions investigated so far were generated by the same transposon family is remarkable and is conceivably due to Galileo's unusual structure and current (or recent) transpositional activity.

  13. Characterization and Diversity of Novel PIF/Harbinger DNA Transposons in Brassica Genomes

    International Nuclear Information System (INIS)

    Nouroz, F.; Noreen, S.; Harrison, H.

    2016-01-01

    Among DNA transposons, PIF/Harbinger is most recently identified superfamily characterized by 3 bp target site duplications (TSDs), flanked by 14-45 bp terminal inverted repeats (TIRs) and displaying DDD or DDE domain displaying transposase. Their autonomous elements contain two open reading frames, ORF1 and ORF2 encoding superfamily specific transposase and DNA-binding domain. Harbinger DNA transposons are recently identified in few plants. In present study, computational and molecular approaches were used for the identification of 8 Harbinger transposons, of which only 2 were complete with putative trans posase, while rest 6 lack transposase and are considered as defective or non-autonomous elements. They ranged in size from 0.5-4 kb with 3 bp TSDs, 15-42 bp TIRs and internal AT richregions. The PCR amplification of Brassica Harbinger transposase revealed diversity and ancient nature of these elements. The amplification polymorphism of some non-autonomous Harbingers showed species specific distribution. Phylogenetic analyses of transposase clustered them into two clades (monocot and dicot) and five sub-clades. The Brassica, Arabidopsis and Malustransposase clustered into genera specific sub-clades; although a lot of homology in transposase was observed. The multiple sequence alignment of Brassica and related transposase showed homology in five conserved blocks. The DD/Sub 35/E triad and sequences showed similarity to already known Pong-like or Arabidopsis ATIS12 Harbinger transposase in contrast to other transposase having DD/Sub 47/E or DD/Sub 48/E motifs. The present study will be helpful in the characterization of Harbingers, their structural diversity in related genera and Harbinger based molecular markers for varietal/lines identifications. (author)

  14. Transposon activation mutagenesis as a screening tool for identifying resistance to cancer therapeutics

    International Nuclear Information System (INIS)

    Chen, Li; Schmidt, Emmett V; Stuart, Lynda; Ohsumi, Toshiro K; Burgess, Shawn; Varshney, Gaurav K; Dastur, Anahita; Borowsky, Mark; Benes, Cyril; Lacy-Hulbert, Adam

    2013-01-01

    The development of resistance to chemotherapies represents a significant barrier to successful cancer treatment. Resistance mechanisms are complex, can involve diverse and often unexpected cellular processes, and can vary with both the underlying genetic lesion and the origin or type of tumor. For these reasons developing experimental strategies that could be used to understand, identify and predict mechanisms of resistance in different malignant cells would be a major advance. Here we describe a gain-of-function forward genetic approach for identifying mechanisms of resistance. This approach uses a modified piggyBac transposon to generate libraries of mutagenized cells, each containing transposon insertions that randomly activate nearby gene expression. Genes of interest are identified using next-gen high-throughput sequencing and barcode multiplexing is used to reduce experimental cost. Using this approach we successfully identify genes involved in paclitaxel resistance in a variety of cancer cell lines, including the multidrug transporter ABCB1, a previously identified major paclitaxel resistance gene. Analysis of co-occurring transposons integration sites in single cell clone allows for the identification of genes that might act cooperatively to produce drug resistance a level of information not accessible using RNAi or ORF expression screening approaches. We have developed a powerful pipeline to systematically discover drug resistance in mammalian cells in vitro. This cost-effective approach can be readily applied to different cell lines, to identify canonical or context specific resistance mechanisms. Its ability to probe complex genetic context and non-coding genomic elements as well as cooperative resistance events makes it a good complement to RNAi or ORF expression based screens

  15. Tc7, a Tc1-hitch hiking transposon in Caenorhabditis elegans.

    OpenAIRE

    Rezsohazy, R; van Luenen, H G; Durbin, R M; Plasterk, R H

    1997-01-01

    We have found a novel transposon in the genome of Caenorhabditis elegans. Tc7 is a 921 bp element, made up of two 345 bp inverted repeats separated by a unique, internal sequence. Tc7 does not contain an open reading frame. The outer 38 bp of the inverted repeat show 36 matches with the outer 38 bp of Tc1. This region of Tc1 contains the Tc1-transposase binding site. Furthermore, Tc7 is flanked by TA dinucleotides, just like Tc1, which presumably correspond to the target duplication generated...

  16. Toward forward genetic screens in malaria-causing parasites using the piggyBac transposon

    Directory of Open Access Journals (Sweden)

    de Koning-Ward Tania F

    2011-03-01

    Full Text Available Abstract The ability to analyze gene function in malaria-causing Plasmodium parasites has received a boost with a recent paper in BMC Genomics that describes a genome-wide mutagenesis system in the rodent malaria species Plasmodium berghei using the transposon piggyBac. This advance holds promise for identifying and validating new targets for intervention against malaria. But further improvements are still needed for the full power of genome-wide molecular genetic screens to be utilized in this organism. See research article: http://www.biomedcentral.com/1471-2164/12/155

  17. Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon

    Directory of Open Access Journals (Sweden)

    Hackett Perry B

    2006-06-01

    Full Text Available Abstract Background Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern. Results Here we report the systematic development and testing of a transposon-based gene-trap system incorporating the doxycycline-repressible Tet-Off (tTA system that is capable of activating the expression of genes under control of a Tet response element (TRE promoter. We demonstrate that the gene trap system is fully functional in vitro by introducing the "gene-trap tTA" vector into human cells by transposition and identifying clones that activate expression of a TRE-luciferase transgene in a doxycycline-dependent manner. In transgenic mice, we mobilize gene-trap tTA vectors, discover parameters that can affect germline mobilization rates, and identify candidate gene insertions to demonstrate the in vivo functionality of the vector system. We further demonstrate that the gene-trap can act as a reporter of endogenous gene expression and it can be coupled with bioluminescent imaging to identify genes with tissue-specific expression patterns. Conclusion Akin to the GAL4/UAS system used in the fly, we have made progress developing a tool for mutating and revealing the expression of mouse genes by generating the tTA transactivator in the presence of a secondary TRE-regulated reporter molecule. A vector like the gene

  18. Transposon mutations in the flagella biosynthetic pathway of the solvent-tolerant Pseudomonas putida S12 result in a decreased expression of solvent efflux genes

    NARCIS (Netherlands)

    Kieboom, J; Bruinenberg, R; Keizer-Gunnink, [No Value; de Bont, JAM

    2001-01-01

    Fourteen solvent-sensitive transposon mutants were generated from the solvent-tolerant Pseudomonas putida strain S12 by applying the TnMOD-KmO mutagenesis system. These mutants were unable to grow in the presence of octanol and toluene. By cloning the region flanking the transposon insertion point a

  19. Facile construction of a random protein domain insertion library using an engineered transposon.

    Science.gov (United States)

    Shah, Vandan; Pierre, Brennal; Kim, Jin Ryoun

    2013-01-15

    Insertional fusion between multiple protein domains represents a novel means of creating integrated functionalities. Currently, there is no robust guideline for selection of insertion sites ensuring the desired functional outcome of insertional fusion. Therefore, construction and testing of random domain insertion libraries, in which a host protein domain is randomly inserted into a guest protein domain, significantly benefit extensive exploration of sequence spaces for insertion sites. Short peptide residues are usually introduced between protein domains to alleviate structural conflicts, and the interdomain linker residues may affect the functional outcome of protein insertion complexes. Unfortunately, optimal control of interdomain linker residues is not always available in conventional methods used to construct random domain insertion libraries. Moreover, most conventional methods employ blunt-end rather than sticky-end ligation between host and guest DNA fragments, thus lowering library construction efficiency. Here, we report the facile construction of random domain insertion libraries using an engineered transposon. We show that random domain insertion with optimal control of interdomain linker residues was possible with our engineered transposon-based method. In addition, our method employs sticky-end rather than blunt-end ligation between host and guest DNA fragments, thus allowing for facile construction of relatively large sized libraries. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Fast and efficient Drosophila melanogaster gene knock-ins using MiMIC transposons.

    Science.gov (United States)

    Vilain, Sven; Vanhauwaert, Roeland; Maes, Ine; Schoovaerts, Nils; Zhou, Lujia; Soukup, Sandra; da Cunha, Raquel; Lauwers, Elsa; Fiers, Mark; Verstreken, Patrik

    2014-10-08

    Modern molecular genetics studies necessitate the manipulation of genes in their endogenous locus, but most of the current methodologies require an inefficient donor-dependent homologous recombination step to locally modify the genome. Here we describe a methodology to efficiently generate Drosophila knock-in alleles by capitalizing on the availability of numerous genomic MiMIC transposon insertions carrying recombinogenic attP sites. Our methodology entails the efficient PhiC31-mediated integration of a recombination cassette flanked by unique I-SceI and/or I-CreI restriction enzyme sites into an attP-site. These restriction enzyme sites allow for double-strand break-mediated removal of unwanted flanking transposon sequences, while leaving the desired genomic modifications or recombination cassettes. As a proof-of-principle, we mutated LRRK, tau, and sky by using different MiMIC elements. We replaced 6 kb of genomic DNA encompassing the tau locus and 35 kb encompassing the sky locus with a recombination cassette that permits easy integration of DNA at these loci and we also generated a functional LRRK(HA) knock in allele. Given that ~92% of the Drosophila genes are located within the vicinity (MiMIC element, our methodology enables the efficient manipulation of nearly every locus in the fruit fly genome without the need for inefficient donor-dependent homologous recombination events. Copyright © 2014 Vilain et al.

  1. MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

    Science.gov (United States)

    Venken, Koen J. T.; Schulze, Karen L.; Haelterman, Nele A.; Pan, Hongling; He, Yuchun; Evans-Holm, Martha; Carlson, Joseph W.; Levis, Robert W.; Spradling, Allan C.; Hoskins, Roger A.; Bellen, Hugo J.

    2011-01-01

    We demonstrate the versatility of a collection of insertions of the transposon Minos mediated integration cassette (MiMIC), in Drosophila melanogaster. MiMIC contains a gene-trap cassette and the yellow+ marker flanked by two inverted bacteriophage ΦC31 attP sites. MiMIC integrates almost at random in the genome to create sites for DNA manipulation. The attP sites allow the replacement of the intervening sequence of the transposon with any other sequence through recombinase mediated cassette exchange (RMCE). We can revert insertions that function as gene traps and cause mutant phenotypes to wild type by RMCE and modify insertions to control GAL4 or QF overexpression systems or perform lineage analysis using the Flp system. Insertions within coding introns can be exchanged with protein-tag cassettes to create fusion proteins to follow protein expression and perform biochemical experiments. The applications of MiMIC vastly extend the Drosophila melanogaster toolkit. PMID:21985007

  2. The BDGP gene disruption project: Single transposon insertions associated with 40 percent of Drosophila genes

    Energy Technology Data Exchange (ETDEWEB)

    Bellen, Hugo J.; Levis, Robert W.; Liao, Guochun; He, Yuchun; Carlson, Joseph W.; Tsang, Garson; Evans-Holm, Martha; Hiesinger, P. Robin; Schulze, Karen L.; Rubin, Gerald M.; Hoskins, Roger A.; Spradling, Allan C.

    2004-01-13

    The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in more than 30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6,300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7,140 lines. It now includes individual lines predicted to disrupt 5,362 of the 13,666 currently annotated Drosophila genes (39 percent). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene mis-expression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.

  3. Identification of novel genes responsible for ethanol and/or thermotolerance by transposon mutagenesis in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyun-Soo [Ewha Womans Univ., Seoul (Korea, Republic of). Dept. of Life Sciences; Kim, Na-Rae [Ewha Womans Univ., Seoul (Korea, Republic of). Div. of Life and Pharmaceutical Sciences; Yang, Jungwoo [Ewha Womans Univ., Seoul (Korea, Republic of). Microbial Resources Research Center; Choi, Wonja [Ewha Womans Univ., Seoul (Korea, Republic of). Dept. of Life Sciences; Ewha Womans Univ., Seoul (Korea, Republic of). Div. of Life and Pharmaceutical Sciences; Ewha Womans Univ., Seoul (Korea, Republic of). Microbial Resources Research Center

    2011-08-15

    Saccharomyces cerevisiae strains tolerant to ethanol and heat stresses are important for industrial ethanol production. In this study, five strains (Tn 1-5) tolerant to up to 15% ethanol were isolated by screening a transposon-mediated mutant library. Two of them displayed tolerance to heat (42 C). The determination of transposon insertion sites and Northern blot analysis identified seven putative genes (CMP2, IMD4, SSK2, PPG1, DLD3, PAM1, and MSN2) and revealed simultaneous down-regulations of CMP2 and IMD4, and SSK2 and PPG1, down-regulation of DLD3, and disruptions of the open reading frame of PAM1 and MSN2, indicating that ethanol and/or heat tolerance can be conferred. Knockout mutants of these seven individual genes were ethanol tolerant and three of them (SSK2, PPG1, and PAM1) were tolerant to heat. Such tolerant phenotypes reverted to sensitive phenotypes by the autologous or overexpression of each gene. Five transposon mutants showed higher ethanol production and grew faster than the control strain when cultured in rich media containing 30% glucose and initial 6% ethanol at 30 C. Of those, two thermotolerant transposon mutants (Tn 2 and Tn 3) exhibited significantly enhanced growth and ethanol production compared to the control at 42 C. The genes identified in this study may provide a basis for the application in developing industrial yeast strains. (orig.)

  4. Molecular characterization of Vulmar1, a complete mariner transposon of sugar beet and diversity of mariner- and En/Spm-like sequences in the genus Beta.

    Science.gov (United States)

    Jacobs, Gunnar; Dechyeva, Daryna; Menzel, Gerhard; Dombrowski, Cora; Schmidt, Thomas

    2004-12-01

    Transposons of the Tc1-mariner superfamily are widespread in eukaryotic genomes. We have isolated the mariner element Vulmar1 from Beta vulgaris L., which is 3909 bp long and bordered by perfect terminal inverted repeats of 32 bp with homology to terminal inverted repeats of transposons from soybean and rice. According to a characteristic amino acid signature, Vulmar1 can be assigned to the DD39D group of mariner transposons. Vulmar1 is flanked by a 5'-TA-3' target site duplication that is typical for mariner transposons. Southern hybridization revealed that mariner-like copies are highly abundant in Beta species, and sequence analysis of 10 transposase fragments from representative species of the four Beta sections revealed an identity between 34% and 100% after conceptual translation. By fluorescent in situ hybridization, Vulmar1 was detected in distal euchromatin as well as in some intercalary and pericentromeric regions of all B. vulgaris chromosomes. In addition, using PCR, we were able to amplify fragments of the transposase gene of En/Spm-like transposons in the genus Beta. En/Spm-like transposase sequences are highly amplified in four Beta sections and showed a considerable degree of conservation (88.5-100%) at the protein level, while the homology to corresponding regions of En/Spm transposons of other plant species ranges from 49.5% to 62.5%. By fluorescent in situ hybridization, En/Spm-like transposon signals of strong intensity were detected on all chromosomes of B. vulgaris.

  5. The maternal-effect, selfish genetic element Medea is associated with a composite Tc1 transposon.

    Science.gov (United States)

    Lorenzen, Marcé D; Gnirke, Andreas; Margolis, Jonathan; Garnes, Jeffrey; Campbell, Margie; Stuart, Jeffrey J; Aggarwal, Rajat; Richards, Stephen; Park, Yoonseong; Beeman, Richard W

    2008-07-22

    Maternal-Effect Dominant Embryonic Arrest ("Medea") factors are selfish nuclear elements that combine maternal-lethal and zygotic-rescue activities to gain a postzygotic survival advantage. We show that Medea(1) activity in Tribolium castaneum is associated with a composite Tc1 transposon inserted just downstream of the neurotransmitter reuptake symporter bloated tubules (blot), whose Drosophila ortholog has both maternal and zygotic functions. The 21.5-kb insertion contains defective copies of elongation initiation factor-3, ATP synthase subunit C, and an RNaseD-related gene, as well as a potentially intact copy of a prokaryotic DUF1703 gene. Sequence comparisons suggest that the current distribution of Medea(1) reflects global emanation after a single transpositional event in recent evolutionary time. The Medea system in Tribolium represents an unusual type of intragenomic conflict and could provide a useful vehicle for driving desirable genes into populations.

  6. Structural and sequence diversity of the transposon Galileo in the Drosophila willistoni genome.

    Science.gov (United States)

    Gonçalves, Juliana W; Valiati, Victor Hugo; Delprat, Alejandra; Valente, Vera L S; Ruiz, Alfredo

    2014-09-13

    Galileo is one of three members of the P superfamily of DNA transposons. It was originally discovered in Drosophila buzzatii, in which three segregating chromosomal inversions were shown to have been generated by ectopic recombination between Galileo copies. Subsequently, Galileo was identified in six of 12 sequenced Drosophila genomes, indicating its widespread distribution within this genus. Galileo is strikingly abundant in Drosophila willistoni, a neotropical species that is highly polymorphic for chromosomal inversions, suggesting a role for this transposon in the evolution of its genome. We carried out a detailed characterization of all Galileo copies present in the D. willistoni genome. A total of 191 copies, including 133 with two terminal inverted repeats (TIRs), were classified according to structure in six groups. The TIRs exhibited remarkable variation in their length and structure compared to the most complete copy. Three copies showed extended TIRs due to internal tandem repeats, the insertion of other transposable elements (TEs), or the incorporation of non-TIR sequences into the TIRs. Phylogenetic analyses of the transposase (TPase)-encoding and TIR segments yielded two divergent clades, which we termed Galileo subfamilies V and W. Target-site duplications (TSDs) in D. willistoni Galileo copies were 7- or 8-bp in length, with the consensus sequence GTATTAC. Analysis of the region around the TSDs revealed a target site motif (TSM) with a 15-bp palindrome that may give rise to a stem-loop secondary structure. There is a remarkable abundance and diversity of Galileo copies in the D. willistoni genome, although no functional copies were found. The TIRs in particular have a dynamic structure and extend in different ways, but their ends (required for transposition) are more conserved than the rest of the element. The D. willistoni genome harbors two Galileo subfamilies (V and W) that diverged ~9 million years ago and may have descended from an ancestral

  7. Transcriptome-wide Identification and Expression Analysis of Brachypodium distachyon Transposons in Response to Viral Infection

    Directory of Open Access Journals (Sweden)

    Tuğba Gürkök

    2017-10-01

    Full Text Available Transposable elements (TEs are the most abundant group of genomic elements in plants that can be found in genic or intergenic regions of their host genomes. Several stimuli such as biotic or abiotic stress have roles in either activating their transcription or transposition. Here the effect of the Panicum mosaic virus (PMV and its satellite virus (SPMV infection on the transposon transcription of the Brachypodium distachyon model plant was investigated. To evaluate the transcription activity of TEs, transcriptomic data of mock and virus inoculated plants were compared. Our results indicate that major components of TEs are retroelements in all RNA-seq libraries. The number of transcribed TEs detected in mock inoculated plants is higher than virus inoculated plants. In comparison with mock inoculated plants 13% of the TEs showed at least two folds alteration upon PMV infection and 21% upon PMV+SPMV infection. Rather than inoculation with PMV alone inoculation with PMV+SPMV together also increased various TE encoding transcripts expressions. MuDR-N78C_OS encoding transcript was strongly up-regulated against both PMV and PMV+SPMV infection. The synergism generated by PMV and SPMV together enhanced TE transcripts expressions than PMV alone. It was observed that viral infection induced the transcriptional activity of several transposons. The results suggest that increased expressions of TEs might have a role in response to biotic stress in B. distachyon. Identification of TEs which are taking part in stress can serve useful information for functional genomics and designing novel breeding strategies in developing stress resistance crops.

  8. A large inversion in the linear chromosome of Streptomyces griseus caused by replicative transposition of a new Tn3 family transposon.

    Science.gov (United States)

    Murata, M; Uchida, T; Yang, Y; Lezhava, A; Kinashi, H

    2011-04-01

    We have comprehensively analyzed the linear chromosomes of Streptomyces griseus mutants constructed and kept in our laboratory. During this study, macrorestriction analysis of AseI and DraI fragments of mutant 402-2 suggested a large chromosomal inversion. The junctions of chromosomal inversion were cloned and sequenced and compared with the corresponding target sequences in the parent strain 2247. Consequently, a transposon-involved mechanism was revealed. Namely, a transposon originally located at the left target site was replicatively transposed to the right target site in an inverted direction, which generated a second copy and at the same time caused a 2.5-Mb chromosomal inversion. The involved transposon named TnSGR was grouped into a new subfamily of the resolvase-encoding Tn3 family transposons based on its gene organization. At the end, terminal diversity of S. griseus chromosomes is discussed by comparing the sequences of strains 2247 and IFO13350.

  9. Salmonella Persistence in Tomatoes Requires a Distinct Set of Metabolic Functions Identified by Transposon Insertion Sequencing

    Science.gov (United States)

    Desai, Prerak; Porwollik, Steffen; Canals, Rocio; Perez, Daniel R.; Chu, Weiping; McClelland, Michael; Teplitski, Max

    2016-01-01

    ABSTRACT Human enteric pathogens, such as Salmonella spp. and verotoxigenic Escherichia coli, are increasingly recognized as causes of gastroenteritis outbreaks associated with the consumption of fruits and vegetables. Persistence in plants represents an important part of the life cycle of these pathogens. The identification of the full complement of Salmonella genes involved in the colonization of the model plant (tomato) was carried out using transposon insertion sequencing analysis. With this approach, 230,000 transposon insertions were screened in tomato pericarps to identify loci with reduction in fitness, followed by validation of the screen results using competition assays of the isogenic mutants against the wild type. A comparison with studies in animals revealed a distinct plant-associated set of genes, which only partially overlaps with the genes required to elicit disease in animals. De novo biosynthesis of amino acids was critical to persistence within tomatoes, while amino acid scavenging was prevalent in animal infections. Fitness reduction of the Salmonella amino acid synthesis mutants was generally more severe in the tomato rin mutant, which hyperaccumulates certain amino acids, suggesting that these nutrients remain unavailable to Salmonella spp. within plants. Salmonella lipopolysaccharide (LPS) was required for persistence in both animals and plants, exemplifying some shared pathogenesis-related mechanisms in animal and plant hosts. Similarly to phytopathogens, Salmonella spp. required biosynthesis of amino acids, LPS, and nucleotides to colonize tomatoes. Overall, however, it appears that while Salmonella shares some strategies with phytopathogens and taps into its animal virulence-related functions, colonization of tomatoes represents a distinct strategy, highlighting this pathogen's flexible metabolism. IMPORTANCE Outbreaks of gastroenteritis caused by human pathogens have been increasingly associated with foods of plant origin, with tomatoes

  10. Time-Resolved Transposon Insertion Sequencing Reveals Genome-Wide Fitness Dynamics during Infection.

    Science.gov (United States)

    Yang, Guanhua; Billings, Gabriel; Hubbard, Troy P; Park, Joseph S; Yin Leung, Ka; Liu, Qin; Davis, Brigid M; Zhang, Yuanxing; Wang, Qiyao; Waldor, Matthew K

    2017-10-03

    Transposon insertion sequencing (TIS) is a powerful high-throughput genetic technique that is transforming functional genomics in prokaryotes, because it enables genome-wide mapping of the determinants of fitness. However, current approaches for analyzing TIS data assume that selective pressures are constant over time and thus do not yield information regarding changes in the genetic requirements for growth in dynamic environments (e.g., during infection). Here, we describe structured analysis of TIS data collected as a time series, termed pattern analysis of conditional essentiality (PACE). From a temporal series of TIS data, PACE derives a quantitative assessment of each mutant's fitness over the course of an experiment and identifies mutants with related fitness profiles. In so doing, PACE circumvents major limitations of existing methodologies, specifically the need for artificial effect size thresholds and enumeration of bacterial population expansion. We used PACE to analyze TIS samples of Edwardsiella piscicida (a fish pathogen) collected over a 2-week infection period from a natural host (the flatfish turbot). PACE uncovered more genes that affect E. piscicida 's fitness in vivo than were detected using a cutoff at a terminal sampling point, and it identified subpopulations of mutants with distinct fitness profiles, one of which informed the design of new live vaccine candidates. Overall, PACE enables efficient mining of time series TIS data and enhances the power and sensitivity of TIS-based analyses. IMPORTANCE Transposon insertion sequencing (TIS) enables genome-wide mapping of the genetic determinants of fitness, typically based on observations at a single sampling point. Here, we move beyond analysis of endpoint TIS data to create a framework for analysis of time series TIS data, termed pattern analysis of conditional essentiality (PACE). We applied PACE to identify genes that contribute to colonization of a natural host by the fish pathogen

  11. Restorer-of-Fertility Mutations Recovered in Transposon-Active Lines of S Male-Sterile Maize

    Directory of Open Access Journals (Sweden)

    Susan Gabay-Laughnan

    2018-01-01

    Full Text Available Mitochondria execute key pathways of central metabolism and serve as cellular sensing and signaling entities, functions that depend upon interactions between mitochondrial and nuclear genetic systems. This is exemplified in cytoplasmic male sterility type S (CMS-S of Zea mays, where novel mitochondrial open reading frames are associated with a pollen collapse phenotype, but nuclear restorer-of-fertility (restorer mutations rescue pollen function. To better understand these genetic interactions, we screened Activator-Dissociation (Ac-Ds, Enhancer/Suppressor-mutator (En/Spm, and Mutator (Mu transposon-active CMS-S stocks to recover new restorer mutants. The frequency of restorer mutations increased in transposon-active stocks compared to transposon-inactive stocks, but most mutants recovered from Ac-Ds and En/Spm stocks were unstable, reverting upon backcrossing to CMS-S inbred lines. However, 10 independent restorer mutations recovered from CMS-S Mu transposon stocks were stable upon backcrossing. Many restorer mutations condition seed-lethal phenotypes that provide a convenient test for allelism. Eight such mutants recovered in this study included one pair of allelic mutations that were also allelic to the previously described rfl2-1 mutant. Targeted analysis of mitochondrial proteins by immunoblot identified two features that consistently distinguished restored CMS-S pollen from comparably staged, normal-cytoplasm, nonmutant pollen: increased abundance of nuclear-encoded alternative oxidase relative to mitochondria-encoded cytochrome oxidase and decreased abundance of mitochondria-encoded ATP synthase subunit 1 compared to nuclear-encoded ATP synthase subunit 2. CMS-S restorer mutants thus revealed a metabolic plasticity in maize pollen, and further study of these mutants will provide new insights into mitochondrial functions that are critical to pollen and seed development.

  12. Increased encapsulated cell biodelivery of nerve growth factor in the brain by transposon-mediated gene transfer

    DEFF Research Database (Denmark)

    Fjord-Larsen, L; Kusk, Poul Henrik; Emerich, D F

    2012-01-01

    transposon expression technology to establish a new clinical grade cell line, NGC0211, with at least 10 times higher NGF production than that of NGC-0295. To test whether encapsulation of this cell line provides a relevant dose escalation step in delivering NGF for treatment of the cognitive decline in AD...... cases correlate with highly improved potency.Gene Therapy advance online publication, 24 November 2011; doi:10.1038/gt.2011.178....

  13. Microbial Fe (III) reduction and hydrogen production by a transposon-mutagenized strain of Pantoea agglomerans BH18

    International Nuclear Information System (INIS)

    Liu, Hongyan; Wang, Guangce

    2015-01-01

    Based on the transposon-mutagenized library of Pantoea agglomerans BH18, mutant screens were conducted to obtain the strain with the highest Fe (III) reduction and hydrogen production. Of these transposon-mutagenized mutants, the mutant strain TB230 was screened for high Fe (III)-reducing efficiency and hydrogen production. The PCR amplification and kanamycin resistance selection results indicated that the transposon insertion of the mutant strain TB230 was stable. Hydrogen production of the mutant strain TB230 was (2.21 ± 0.34) mol H 2 /mol glucose, which increased hydrogen production by over 40% compared with that of the wild type strain. The accumulation concentration of Fe (II) in the medium of the mutant strain TB230 with Fe (OH) 3 as the sole electron acceptor was (7.39 ± 0.49) mmol/l, which was approximately 3-fold greater than that of the wild type strain. The mutant strain TB230 showed high Fe (III)-reducing activity and hydrogen production by adopting glucose and pyruvate as the carbon source. In addition, the mutant strain TB230 was capable of Fe (III) reduction and hydrogen production under fresh or marine conditions. This result indicates that the mutant strain with high microbial Fe (III) reduction and hydrogen production is beneficial for the improvement of anaerobic performance. - Highlights: • The mutant strain TB230 was a transposon-mutagenized strain of Pantoea agglomerans BH18. • Strain TB230 was screened for high Fe (III)-reducing efficiency and hydrogen production. • H 2 yield and Fe (III)-reducing activity were 2.21 ± 0.34 and 7.39 ± 0.49 in marine condition. • Strain TB230 was capable of Fe (III) reduction and hydrogen production in fresh or marine condition

  14. IS30-related transposon mediated insertional inactivation of bile salt hydrolase (bsh1) gene of Lactobacillus plantarum strain Lp20.

    Science.gov (United States)

    Kumar, Rajesh; Grover, Sunita; Kaushik, Jai K; Batish, Virender Kumar

    2014-01-01

    Lactobacillus plantarum is a flexible and versatile microorganism that inhabits a variety of niches, and its genome may express up to four bsh genes to maximize its survival in the mammalian gut. However, the ecological significance of multiple bsh genes in L. plantarum is still not clearly understood. Hence, this study demonstrated the disruption of bile salt hydrolase (bsh1) gene due to the insertion of a transposable element in L. plantarum Lp20 - a wild strain of human fecal origin. Surprisingly, L. plantarum strain Lp20 produced a ∼2.0 kb bsh1 amplicon against the normal size (∼1.0 kb) bsh1 amplicon of Bsh(+)L. plantarum Lp21. Strain Lp20 exhibited minimal Bsh activity in spite of having intact bsh2, bsh3 and bsh4 genes in its genome and hence had a Bsh(-) phenotype. Cloning and sequence characterization of Lp20 bsh1 gene predicted four individual open reading frames (ORFs) within this region. BLAST analysis of ORF1 and ORF2 revealed significant sequence similarity to the L. plantarum bsh1 gene while ORF3 and ORF4 showed high sequence homology to IS30-family transposases. Since, IS30-related transposon element was inserted within Lp20 bsh1 gene in reverse orientation (3'-5'), it introduced several stop codons and disrupted the protein reading frames of both Bsh1 and transposase. Inverted terminal repeats (GGCAGATTG) of transposon, mediated its insertion at 255-263 nt and 1301-1309 nt positions of Lp20 bsh1 gene. In conclusion, insertion of IS30 related-transposon within the bsh1 gene sequence of L. plantarum strain Lp20 demolished the integrity and functionality of Bsh1 enzyme. Additionally, this transposon DNA sequence remains active among various Lactobacillus spp. and hence harbors the potential to be explored in the development of efficient insertion mutagenesis system. Copyright © 2013 Elsevier GmbH. All rights reserved.

  15. TED, an Autonomous and Rare Maize Transposon of the Mutator Superfamily with a High Gametophytic Excision Frequency[W

    Science.gov (United States)

    Li, Yubin; Harris, Linda; Dooner, Hugo K.

    2013-01-01

    Mutator (Mu) elements, one of the most diverse superfamilies of DNA transposons, are found in all eukaryotic kingdoms, but are particularly numerous in plants. Most of the present knowledge on the transposition behavior of this superfamily comes from studies of the maize (Zea mays) Mu elements, whose transposition is mediated by the autonomous Mutator-Don Robertson (MuDR) element. Here, we describe the maize element TED (for Transposon Ellen Dempsey), an autonomous cousin that differs significantly from MuDR. Element excision and reinsertion appear to require both proteins encoded by MuDR, but only the single protein encoded by TED. Germinal excisions, rare with MuDR, are common with TED, but arise in one of the mitotic divisions of the gametophyte, rather than at meiosis. Instead, transposition-deficient elements arise at meiosis, suggesting that the double-strand breaks produced by element excision are repaired differently in mitosis and meiosis. Unlike MuDR, TED is a very low-copy transposon whose number and activity do not undergo dramatic changes upon inbreeding or outcrossing. Like MuDR, TED transposes mostly to unlinked sites and can form circular transposition products. Sequences closer to TED than to MuDR were detected only in the grasses, suggesting a rather recent evolutionary split from a common ancestor. PMID:24038653

  16. TED, an autonomous and rare maize transposon of the mutator superfamily with a high gametophytic excision frequency.

    Science.gov (United States)

    Li, Yubin; Harris, Linda; Dooner, Hugo K

    2013-09-01

    Mutator (Mu) elements, one of the most diverse superfamilies of DNA transposons, are found in all eukaryotic kingdoms, but are particularly numerous in plants. Most of the present knowledge on the transposition behavior of this superfamily comes from studies of the maize (Zea mays) Mu elements, whose transposition is mediated by the autonomous Mutator-Don Robertson (MuDR) element. Here, we describe the maize element TED (for Transposon Ellen Dempsey), an autonomous cousin that differs significantly from MuDR. Element excision and reinsertion appear to require both proteins encoded by MuDR, but only the single protein encoded by TED. Germinal excisions, rare with MuDR, are common with TED, but arise in one of the mitotic divisions of the gametophyte, rather than at meiosis. Instead, transposition-deficient elements arise at meiosis, suggesting that the double-strand breaks produced by element excision are repaired differently in mitosis and meiosis. Unlike MuDR, TED is a very low-copy transposon whose number and activity do not undergo dramatic changes upon inbreeding or outcrossing. Like MuDR, TED transposes mostly to unlinked sites and can form circular transposition products. Sequences closer to TED than to MuDR were detected only in the grasses, suggesting a rather recent evolutionary split from a common ancestor.

  17. Identification of genes potentially involved in solute stress response in Sphingomonas wittichii RW1 by transposon mutant recovery

    Directory of Open Access Journals (Sweden)

    Edith eCoronado

    2014-11-01

    Full Text Available The term water stress refers to the effects of low water availability on microbial growth and physiology. Water availability has been proposed as a major constraint for the use of microorganisms in contaminated sites with the purpose of bioremediation. Sphingomonas wittichii RW1 is a bacterium capable of degrading the xenobiotic compounds dibenzofuran and dibenzo-p-dioxin, and has potential to be used for targeted bioremediation. The aim of the current work was to identify genes implicated in water stress in RW1 by means of transposon mutagenesis and mutant growth experiments. Conditions of low water potential were mimicked by adding NaCl to the growth media. Three different mutant selection or separation method were tested, which, however recovered different mutants. Recovered transposon mutants with poorer growth under salt-induced water stress carried insertions in genes involved in proline and glutamate biosynthesis, and further in a gene putatively involved in aromatic compound catabolism. Transposon mutants growing poorer on medium with lowered water potential also included ones that had insertions in genes involved in more general functions such as transcriptional regulation, elongation factor, cell division protein, RNA polymerase β or an aconitase.

  18. Chick derived induced pluripotent stem cells by the poly-cistronic transposon with enhanced transcriptional activity.

    Science.gov (United States)

    Katayama, Masafumi; Hirayama, Takashi; Tani, Tetsuya; Nishimori, Katsuhiko; Onuma, Manabu; Fukuda, Tomokazu

    2018-02-01

    Induced pluripotent stem (iPS) cell technology lead terminally differentiated cells into the pluripotent stem cells through the expression of defined reprogramming factors. Although, iPS cells have been established in a number of mammalian species, including mouse, human, and monkey, studies on iPS cells in avian species are still very limited. To establish chick iPS cells, six factors were used within the poly-cistronic reprogramming vector (PB-R6F), containing M3O (MyoD derived transactivation domain fused with Oct3/4), Sox2, Klf4, c-Myc, Lin28, and Nanog. The PB-R6F derived iPS cells were alkaline-phosphatase and SSEA-1 positive, which are markers of pluripotency. Elevated levels of endogenous Oct3/4 and Nanog genes were detected in the established iPS cells, suggesting the activation of the FGF signaling pathway is critical for the pluripotent status. Histological analysis of teratoma revealed that the established chick iPS cells have differentiation ability into three-germ-layer derived tissues. This is the first report of establishment of avian derived iPS cells with a single poly-cistronic transposon based expression system. The establishment of avian derived iPS cells could contribute to the genetic conservation and modification of avian species. © 2017 Wiley Periodicals, Inc.

  19. Drosophila transposon insertions as unknowns for structured inquiry recombination mapping exercises in an undergraduate genetics course.

    Science.gov (United States)

    Marcus, Jeffrey M; Hughes, Tia M

    2009-06-01

    Structured inquiry approaches, in which students receive a Drosophila strain of unknown genotype to analyze and map the constituent mutations, are a common feature of many genetics teaching laboratories. The required crosses frustrate many students because they are aware that they are participating in a fundamentally trivial exercise, as the map locations of the genes are already established and have been recalculated thousands of times by generations of students. We modified the traditional structured inquiry approach to include a novel research experience for the students in our undergraduate genetics laboratories. Students conducted crosses with Drosophila strains carrying P[lacW] transposon insertions in genes without documented recombination map positions, representing a large number of unique, but equivalent genetic unknowns. Using the eye color phenotypes associated with the inserts as visible markers, it is straightforward to calculate recombination map positions for the interrupted loci. Collectively, our students mapped 95 genetic loci on chromosomes 2 and 3. In most cases, the calculated 95% confidence interval for meiotic map location overlapped with the predicted map position based on cytology. The research experience evoked positive student responses and helped students better understand the nature of scientific research for little additional cost or instructor effort.

  20. Transposon mutagenesis reveals differential pathogenesis of Ralstonia solanacearum on tomato and Arabidopsis.

    Science.gov (United States)

    Lin, Yu-Mei; Chou, I-Chun; Wang, Jaw-Fen; Ho, Fang-I; Chu, Yu-Ju; Huang, Pei-Cheng; Lu, Der-Kang; Shen, Hwei-Ling; Elbaz, Mounira; Huang, Shu-Mei; Cheng, Chiu-Ping

    2008-09-01

    Ralstonia solanacearum causes a deadly wilting disease on a wide range of crops. To elucidate pathogenesis of this bacterium in different host plants, we set out to identify R. solanacearum genes involved in pathogenesis by screening random transposon insertion mutants of a highly virulent strain, Pss190, on tomato and Arabidopsis thaliana. Mutants exhibiting various decreased virulence levels on these two hosts were identified. Sequence analysis showed that most, but not all, of the identified pathogenesis genes are conserved among distinct R. solanacearum strains. A few of the disrupted loci were not reported previously as being involved in R. solanacearum pathogenesis. Notably, a group of mutants exhibited differential pathogenesis on tomato and Arabidopsis. These results were confirmed by characterizing allelic mutants in one other R. solanacearum strain of the same phylotype. The significantly decreased mutants' colonization in Arabidopsis was found to be correlated with differential pathogenesis on these two plants. Differential requirement of virulence genes suggests adaptation of this bacterium in different host environments. Together, this study reveals commonalities and differences of R. solanacearum pathogenesis on single solanaceous and nonsolanaceous hosts, and provides important new insights into interactions between R. solanacearum and different host plants.

  1. Time-Resolved Transposon Insertion Sequencing Reveals Genome-Wide Fitness Dynamics during Infection

    Directory of Open Access Journals (Sweden)

    Guanhua Yang

    2017-10-01

    Full Text Available Transposon insertion sequencing (TIS is a powerful high-throughput genetic technique that is transforming functional genomics in prokaryotes, because it enables genome-wide mapping of the determinants of fitness. However, current approaches for analyzing TIS data assume that selective pressures are constant over time and thus do not yield information regarding changes in the genetic requirements for growth in dynamic environments (e.g., during infection. Here, we describe structured analysis of TIS data collected as a time series, termed pattern analysis of conditional essentiality (PACE. From a temporal series of TIS data, PACE derives a quantitative assessment of each mutant’s fitness over the course of an experiment and identifies mutants with related fitness profiles. In so doing, PACE circumvents major limitations of existing methodologies, specifically the need for artificial effect size thresholds and enumeration of bacterial population expansion. We used PACE to analyze TIS samples of Edwardsiella piscicida (a fish pathogen collected over a 2-week infection period from a natural host (the flatfish turbot. PACE uncovered more genes that affect E. piscicida’s fitness in vivo than were detected using a cutoff at a terminal sampling point, and it identified subpopulations of mutants with distinct fitness profiles, one of which informed the design of new live vaccine candidates. Overall, PACE enables efficient mining of time series TIS data and enhances the power and sensitivity of TIS-based analyses.

  2. Development of novel metabolite-responsive transcription factors via transposon-mediated protein fusion.

    Science.gov (United States)

    Younger, Andrew K D; Su, Peter Y; Shepard, Andrea J; Udani, Shreya V; Cybulski, Thaddeus R; Tyo, Keith E J; Leonard, Joshua N

    2018-02-01

    Naturally evolved metabolite-responsive biosensors enable applications in metabolic engineering, ranging from screening large genetic libraries to dynamically regulating biosynthetic pathways. However, there are many metabolites for which a natural biosensor does not exist. To address this need, we developed a general method for converting metabolite-binding proteins into metabolite-responsive transcription factors-Biosensor Engineering by Random Domain Insertion (BERDI). This approach takes advantage of an in vitro transposon insertion reaction to generate all possible insertions of a DNA-binding domain into a metabolite-binding protein, followed by fluorescence activated cell sorting to isolate functional biosensors. To develop and evaluate the BERDI method, we generated a library of candidate biosensors in which a zinc finger DNA-binding domain was inserted into maltose binding protein, which served as a model well-studied metabolite-binding protein. Library diversity was characterized by several methods, a selection scheme was deployed, and ultimately several distinct and functional maltose-responsive transcriptional biosensors were identified. We hypothesize that the BERDI method comprises a generalizable strategy that may ultimately be applied to convert a wide range of metabolite-binding proteins into novel biosensors for applications in metabolic engineering and synthetic biology. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  3. A LTR copia retrotransposon and Mutator transposons interrupt Pgip genes in cultivated and wild wheats.

    Science.gov (United States)

    Di Giovanni, Michela; Cenci, Alberto; Janni, Michela; D'Ovidio, Renato

    2008-04-01

    Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defence. Wheat pgip genes have been isolated from the B (Tapgip1) and D (Tapgip2) genomes, and now we report the identification of pgip genes from the A genomes of wild and cultivated wheats. By Southern blots and sequence analysis of BAC clones we demonstrated that wheat contains a single copy pgip gene per genome and the one from the A genome, pgip3, is inactivated by the insertion of a long terminal repeat copia retrotranspon within the fourth LRR. We demonstrated also that this retrotransposon insertion is present in Triticum urartu and all the polyploidy wheats assayed, but is absent in T. monococcum (Tmpgip3), suggesting that this insertion took place after the divergence between T. monococcum and T. urartu, but before the formation of the polyploid wheats. We identified also two independent insertion events of new Class II transposable elements, Vacuna, belonging to the Mutator superfamily, that interrupted the Tdipgip1 gene of T. turgidum ssp. dicoccoides. The occurrence of these transposons within the coding region of Tdipgip1 facilitated the mapping of the Pgip locus in the pericentric region of the short arm of chromosome group 7. We speculate that the inactivation of pgip genes are tolerated because of redundancy of PGIP activities in the wheat genome.

  4. Moving through the stressed genome: Emerging regulatory roles for transposons in plant stress tolerance

    Directory of Open Access Journals (Sweden)

    Negi Pooja

    2016-10-01

    Full Text Available The recognition of a positive correlation between organism genome size with its transposable element (TE content, represents a key discovery of the field of genome biology. Considerable evidence accumulated since then suggests the involvement of TEs in genome structure, evolution and function. The global genome reorganization brought about by transposon activity might play an adaptive/regulatory role in the host response to environmental challenges, reminiscent of McClintock’s original ’Controlling Element’ hypothesis. This regulatory aspect of TEs is also garnering support in light of the recent evidences which project TEs as distributed genomic control modules. According to this view, TEs are capable of actively reprogramming host genes circuits and ultimately fine-tuning the host response to specific environmental stimuli. Moreover, the stress-induced changes in epigenetic status of TE activity may allow TEs to propagate their stress responsive elements to host genes; the resulting genome fluidity can permit phenotypic plasticity and adaptation to stress. Given their predominating presence in the plant genomes, nested organization in the genic regions and potential regulatory role in stress response, TEs hold unexplored potential for crop improvement programs. This review intends to present the current information about the roles played by TEs in plant genome organization, evolution and function, and highlight the regulatory mechanisms in plant stress responses. We will also briefly discuss the connection between TE activity, host epigenetic response and phenotypic plasticity as a critical link for traversing the translational bridge from a purely basic study of TEs, to the applied field of stress adaptation and crop improvement.

  5. Moving through the Stressed Genome: Emerging Regulatory Roles for Transposons in Plant Stress Response.

    Science.gov (United States)

    Negi, Pooja; Rai, Archana N; Suprasanna, Penna

    2016-01-01

    The recognition of a positive correlation between organism genome size with its transposable element (TE) content, represents a key discovery of the field of genome biology. Considerable evidence accumulated since then suggests the involvement of TEs in genome structure, evolution and function. The global genome reorganization brought about by transposon activity might play an adaptive/regulatory role in the host response to environmental challenges, reminiscent of McClintock's original 'Controlling Element' hypothesis. This regulatory aspect of TEs is also garnering support in light of the recent evidences, which project TEs as "distributed genomic control modules." According to this view, TEs are capable of actively reprogramming host genes circuits and ultimately fine-tuning the host response to specific environmental stimuli. Moreover, the stress-induced changes in epigenetic status of TE activity may allow TEs to propagate their stress responsive elements to host genes; the resulting genome fluidity can permit phenotypic plasticity and adaptation to stress. Given their predominating presence in the plant genomes, nested organization in the genic regions and potential regulatory role in stress response, TEs hold unexplored potential for crop improvement programs. This review intends to present the current information about the roles played by TEs in plant genome organization, evolution, and function and highlight the regulatory mechanisms in plant stress responses. We will also briefly discuss the connection between TE activity, host epigenetic response and phenotypic plasticity as a critical link for traversing the translational bridge from a purely basic study of TEs, to the applied field of stress adaptation and crop improvement.

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

    Science.gov (United States)

    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.

  7. In silico characterization of microsatellites in Eucalyptus spp.: abundance, length variation and transposon associations

    Directory of Open Access Journals (Sweden)

    Edenilson Rabello

    2005-01-01

    Full Text Available This study assessed the abundance of microsatellites, or simple sequence repeats (SSR, in 19 Eucalyptus EST libraries from FORESTs, containing cDNA sequences from five species: E. grandis, E. globulus, E. saligna, E. urophylla and E. camaldulensis. Overall, a total of 11,534 SSRs and 8,447 SSR-containing sequences (25.5% of total ESTs were identified, with an average of 1 SSR/2.5 kb when considering all motifs and 1 SSR/3.1 kb when mononucleotides were not included. Dimeric repeats were the most abundant (41.03%, followed by trimerics (36.11% and monomerics (19.59%. The most frequent motifs were A/T (87.24% for monomerics, AG/CT (94.44% for dimerics, CCG/CGG (37.87% for trimerics, AAGG/CCTT (18.75% for tetramerics, AGAGG/CCTCT (14.04% for pentamerics and ACGGCG/CGCCGT (6.30% for hexamerics. According to sequence length, Class II or potentially variable markers were the most commonly found, followed by Class III. Two sequences presented high similarity to previously published Eucalyptus sequences from the NCBI database, EMBRA_72 and EMBRA_122. Local blastn search for transposons did not reveal the presence of any transposable elements with a cut-off value of 10-50. The large number of microsatellites identified will contribute to the refinement of marker-assisted mapping and to the discovery of novel markers for virtually all genes of economic interest.

  8. Transposon mutagenesis identifies novel genes associated with Staphylococcus aureus persister formation

    Directory of Open Access Journals (Sweden)

    Wang ewenjie

    2015-12-01

    Full Text Available Pathogenic bacterial persisters are responsible for the recalcitrance of chronic and persistent infections to antimicrobial therapy. Although the mechanisms of persister formation and survival have been widely studied in Escherichia coli, persistence mechanisms in S. aureus remain largely unknown. Here, we screened a transposon mutant library of a clinical methicillin-resistant Staphylococcus aureus(MRSA)strain, USA500 (ST8, under antibiotic pressure and identified 13 genes whose insertion mutations resulted in a defect in persistence. These candidate genes were further confirmed by evaluating the survival of the mutants upon exposure to levofloxacin and several other stress conditions. We found 13 insertion mutants with significantly lower persister numbers under several stress conditions, including sdhA, sdhB, ureG, mnhG1, fbaA, ctaB, clpX, parE, HOU_0223, HOU_0587, HOU_2091, HOU_2315 and HOU_2346, which mapped into pathways of oxidative phosphorylation, TCA cycle, glycolysis, cell cycle and ABC transporters, suggesting that these genes and pathways may play an important role in persister formation and survival. The newly constructed knockout strains of ureG, sdhA and sdhB and their complemented strains were also tested for defect in persisters following exposure to levofloxacin and several other stress conditions. The results from these experiments were consistent with the screening results, which indicated that deletion of these genes in MRSA USA500 leads to persister defect. These findings provide novel insights into the mechanisms of persister formation and survival in S. aureus and offer new targets for the development of persister-directed antibiotics for the improved treatment of chronic and persistent infections.

  9. Analysis of transposons and repeat composition of the sunflower (Helianthus annuus L.) genome.

    Science.gov (United States)

    Cavallini, Andrea; Natali, Lucia; Zuccolo, Andrea; Giordani, Tommaso; Jurman, Irena; Ferrillo, Veronica; Vitacolonna, Nicola; Sarri, Vania; Cattonaro, Federica; Ceccarelli, Marilena; Cionini, Pier Giorgio; Morgante, Michele

    2010-02-01

    A sample-sequencing strategy combined with slot-blot hybridization and FISH was used to study the composition of the repetitive component of the sunflower genome. One thousand six hundred thirty-eight sequences for a total of 954,517 bp were analyzed. The fraction of sequences that can be classified as repetitive using computational and hybridization approaches amounts to 62% in total. Almost two thirds remain as yet uncharacterized in nature. Of those characterized, most belong to the gypsy superfamily of LTR-retrotransposons. Unlike in other species, where single families can account for large fractions of the genome, it appears that no transposon family has been amplified to very high levels in sunflower. All other known classes of transposable elements were also found. One family of unknown nature (contig 61) was the most repeated in the sunflower genome. The evolution of the repetitive component in the Helianthus genus and in other Asteraceae was studied by comparative analysis of the hybridization of total genomic DNAs from these species to the sunflower small-insert library and compared to gene-based phylogeny. Very little similarity is observed between Helianthus species and two related Asteraceae species outside of the genus. Most repetitive elements are similar in annual and perennial Helianthus species indicating that sequence amplification largely predates such divergence. Gypsy-like elements are more represented in the annuals than in the perennials, while copia-like elements are similarly represented, attesting a different amplification history of the two superfamilies of LTR-retrotransposons in the Helianthus genus.

  10. Moving through the Stressed Genome: Emerging Regulatory Roles for Transposons in Plant Stress Response

    Science.gov (United States)

    Negi, Pooja; Rai, Archana N.; Suprasanna, Penna

    2016-01-01

    The recognition of a positive correlation between organism genome size with its transposable element (TE) content, represents a key discovery of the field of genome biology. Considerable evidence accumulated since then suggests the involvement of TEs in genome structure, evolution and function. The global genome reorganization brought about by transposon activity might play an adaptive/regulatory role in the host response to environmental challenges, reminiscent of McClintock's original ‘Controlling Element’ hypothesis. This regulatory aspect of TEs is also garnering support in light of the recent evidences, which project TEs as “distributed genomic control modules.” According to this view, TEs are capable of actively reprogramming host genes circuits and ultimately fine-tuning the host response to specific environmental stimuli. Moreover, the stress-induced changes in epigenetic status of TE activity may allow TEs to propagate their stress responsive elements to host genes; the resulting genome fluidity can permit phenotypic plasticity and adaptation to stress. Given their predominating presence in the plant genomes, nested organization in the genic regions and potential regulatory role in stress response, TEs hold unexplored potential for crop improvement programs. This review intends to present the current information about the roles played by TEs in plant genome organization, evolution, and function and highlight the regulatory mechanisms in plant stress responses. We will also briefly discuss the connection between TE activity, host epigenetic response and phenotypic plasticity as a critical link for traversing the translational bridge from a purely basic study of TEs, to the applied field of stress adaptation and crop improvement. PMID:27777577

  11. Rescue of mutant fitness defects using in vitro reconstituted designer transposons in Mycoplasma mycoides

    Directory of Open Access Journals (Sweden)

    Bogumil Jacek Karas

    2014-07-01

    Full Text Available With only hundreds of genes contained within their genomes, mycoplasmas have become model organisms for precise understanding of cellular processes, as well as platform organisms for predictable engineering of microbial functions for mission-critical applications. Despite the availability of whole genome writing in Mycoplasma mycoides, some traditional methods for genetic engineering are underdeveloped in mycoplasmas. Here we demonstrate two facile transposon-mediated approaches for introducing genes into the synthetic cell based on M. mycoides. The marker-less approach involves preparing a fragment containing only a small genomic region of interest with flanking transposase-binding sites, followed by in vitro transposase loading and introduction into the cells. The marker-driven approach involves cloning an open reading frame (ORF of interest into a vector containing a marker for mycoplasma transformation, as well as sites for transposase loading and random genomic integration. An innovative feature of this construct is to use a single promoter to express the transformation marker and the introduced ORF. The marker-driven approach can be conveniently applied to any exogenous or synthetic gene without any information on the effect of the gene on the strain, whereas the marker-less approach requires that the fragment has a recognizable effect. Using the marker-less method, we found that a region containing the nusG gene rescues a slow growth phenotype of a strain containing a larger deletion encompassing this gene. Using the marker-driven approach, we better defined this finding, thereby establishing that nusG is required for a normal growth rate in synthetic M. mycoides. These methods are suitable for complementation tests to identify genes responsible for assorted functions lacking in deletion mutants. These approaches are also expected to facilitate rapid testing of various natural and engineered genes or gene clusters from numerous sources in

  12. Bioreactor scale up and protein product quality characterization of piggyBac transposon derived CHO pools.

    Science.gov (United States)

    Rajendra, Yashas; Balasubramanian, Sowmya; Peery, Robert B; Swartling, James R; McCracken, Neil A; Norris, Dawn L; Frye, Christopher C; Barnard, Gavin C

    2017-03-01

    Chinese hamster ovary (CHO) cells remain the most popular host for the production of biopharmaceutical drugs, particularly monoclonal antibodies (mAbs), bispecific antibodies, and Fc-fusion proteins. Creating and characterizing the stable CHO clonally-derived cell lines (CDCLs) needed to manufacture these therapeutic proteins is a lengthy and laborious process. Therefore, CHO pools have increasingly been used to rapidly produce protein to support and enable preclinical drug development. We recently described the generation of CHO pools yielding mAb titers as high as 7.6 g/L in a 16 day bioprocess using piggyBac transposon-mediated gene integration. In this study, we wanted to understand why the piggyBac pool titers were significantly higher (2-10 fold) than the control CHO pools. Higher titers were the result of a combination of increased average gene copy number, significantly higher messenger RNA levels and the homogeneity (i.e. less diverse population distribution) of the piggyBac pools, relative to the control pools. In order to validate the use of piggyBac pools to support preclinical drug development, we then performed an in-depth product quality analysis of purified protein. The product quality of protein obtained from the piggyBac pools was very similar to the product quality profile of protein obtained from the control pools. Finally, we demonstrated the scalability of these pools from shake flasks to 36L bioreactors. Overall, these results suggest that gram quantities of therapeutic protein can be rapidly obtained from piggyBac CHO pools without significantly changing product quality attributes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:534-540, 2017. © 2017 American Institute of Chemical Engineers.

  13. The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

    Science.gov (United States)

    Tsukahara, T; Iwase, N; Kawakami, K; Iwasaki, M; Yamamoto, C; Ohmine, K; Uchibori, R; Teruya, T; Ido, H; Saga, Y; Urabe, M; Mizukami, H; Kume, A; Nakamura, M; Brentjens, R; Ozawa, K

    2015-02-01

    Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

  14. piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART for genetic screens in mice.

    Directory of Open Access Journals (Sweden)

    Sean F Landrette

    Full Text Available Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.

  15. Comprehensive MALDI-TOF biotyping of the non-redundant Harvard Pseudomonas aeruginosa PA14 transposon insertion mutant library.

    Science.gov (United States)

    Oumeraci, Tonio; Jensen, Vanessa; Talbot, Steven R; Hofmann, Winfried; Kostrzewa, Markus; Schlegelberger, Brigitte; von Neuhoff, Nils; Häussler, Susanne

    2015-01-01

    Pseudomonas aeruginosa is a gram-negative bacterium that is ubiquitously present in the aerobic biosphere. As an antibiotic-resistant facultative pathogen, it is a major cause of hospital-acquired infections. Its rapid and accurate identification is crucial in clinical and therapeutic environments. In a large-scale MALDI-TOF mass spectrometry-based screen of the Harvard transposon insertion mutant library of P. aeruginosa strain PA14, intact-cell proteome profile spectra of 5547 PA14 transposon mutants exhibiting a plethora of different phenotypes were acquired and analyzed. Of all P. aeruginosa PA14 mutant profiles 99.7% were correctly identified as P. aeruginosa with the Biotyper software on the species level. On the strain level, 99.99% of the profiles were mapped to five different individual P. aeruginosa Biotyper database entries. A principal component analysis-based approach was used to determine the most important discriminatory mass features between these Biotyper groups. Although technical replicas were consistently categorized to specific Biotyper groups in 94.2% of the mutant profiles, biological replicas were not, indicating that the distinct proteotypes are affected by growth conditions. The PA14 mutant profile collection presented here constitutes the largest coherent P. aeruginosa MALDI-TOF spectral dataset publicly available today. Transposon insertions in thousands of different P. aeruginosa genes did not affect species identification from MALDI-TOF mass spectra, clearly demonstrating the robustness of the approach. However, the assignment of the individual spectra to sub-groups proved to be non-consistent in biological replicas, indicating that the differentiation between biotyper groups in this nosocomial pathogen is unassured.

  16. PLE-wu, a new member of piggyBac transposon family from insect, is active in mammalian cells.

    Science.gov (United States)

    Wu, Chunxiao; Wang, Shu

    2014-10-01

    piggyBac, a highly active transposon in insect and mammalian cells, is a very useful tool in genome manipulation. A new piggyBac-like element (PLE), named PLE-wu, was identified from a mutant baculovirus cultured in sf9 insect cells. This new transposon is 2931 bp in length and encodes two active forms of transposase, a 708-amino acid-long transposase and a short 576-residue-long transposase translated from a downstream in-frame initiation codon. PLE-wu has asymmetric terminal structures, containing 6-bp inverted terminal repeats, 32-bp imperfect inverted and direct sub-terminal repeats. Similar to piggyBac, PLE-wu exhibits traceless excision activity in both insect and mammalian cells, restoring the original TTAA target sequence upon excision. It also retains the insertion activity in mammalian cells with a plasmid to chromosome transposition rate about 10-fold higher than random integration. Plasmid rescue assays revealed that the TTAA target sequence was duplicated at the junctions of the insertion site. Deletion of the terminal sequences including the sub-terminal repeats decreased the transposition activity of the 708-residue-long transposase, while the transposition activity of the short form of transposase was not affected. With its low sequence similarity to piggyBac, PLE-wu will contribute to the understanding the mechanism of PLE transposition, as well as design of new transposon systems with higher activity. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  17. Isolation of bacterial extrachromosomal DNA from human dental plaque associated with periodontal disease, using transposon-aided capture (TRACA).

    Science.gov (United States)

    Warburton, Philip J; Allan, Elaine; Hunter, Stephanie; Ward, John; Booth, Veronica; Wade, William G; Mullany, Peter

    2011-11-01

    The human oral cavity is host to a complex microbial community estimated to comprise >700 bacterial species, of which at least half are thought to be not yet cultivable in vitro. To investigate the plasmids present in this community, we used a transposon-aided capture system, which allowed the isolation of plasmids from human oral supra- and subgingival plaque samples. Thirty-two novel plasmids and a circular molecule that could be an integrase-generated circular intermediate were isolated. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  18. Transposons and integrons in colistin-resistant clones of Klebsiella pneumoniae and Acinetobacter baumannii with epidemic or sporadic behaviour.

    Science.gov (United States)

    Arduino, Sonia M; Quiroga, María Paula; Ramírez, María Soledad; Merkier, Andrea Karina; Errecalde, Laura; Di Martino, Ana; Smayevsky, Jorgelina; Kaufman, Sara; Centrón, Daniela

    2012-10-01

    Multiple transposons, integrons and carbapenemases were found in Klebsiella pneumoniae colistin-resistant isolates as well as a genomic resistance island of the AbaR type in Acinetobacter baumannii colistin-resistant isolates from different hospitals from Buenos Aires City. PFGE analysis showed a polyclonal dissemination of antimicrobial resistance mechanisms among K. pneumoniae isolates, while in A. baumannii isolates the epidemic clone 1 from South America was found. Resistance determinants associated with horizontal gene transfer are contributing to the evolution to pandrug resistance in both epidemic and sporadic clones.

  19. ZBED6, a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth

    DEFF Research Database (Denmark)

    Markljung, Ellen; Jiang, Lin; Jaffe, Jacob D

    2009-01-01

    and find that the protein, named ZBED6, is previously unknown, specific for placental mammals, and derived from an exapted DNA transposon. Silencing of Zbed6 in mouse C2C12 myoblasts affected Igf2 expression, cell proliferation, wound healing, and myotube formation. Chromatin immunoprecipitation (Ch......, including development and transcriptional regulation. The phenotypic effects in mutant pigs and ZBED6-silenced C2C12 myoblasts, the extreme sequence conservation, its nucleolar localization, the broad tissue distribution, and the many target genes with essential biological functions suggest that ZBED6...... is an important transcription factor in placental mammals, affecting development, cell proliferation, and growth....

  20. TAPDANCE: An automated tool to identify and annotate transposon insertion CISs and associations between CISs from next generation sequence data

    Directory of Open Access Journals (Sweden)

    Sarver Aaron L

    2012-06-01

    Full Text Available Abstract Background Next generation sequencing approaches applied to the analyses of transposon insertion junction fragments generated in high throughput forward genetic screens has created the need for clear informatics and statistical approaches to deal with the massive amount of data currently being generated. Previous approaches utilized to 1 map junction fragments within the genome and 2 identify Common Insertion Sites (CISs within the genome are not practical due to the volume of data generated by current sequencing technologies. Previous approaches applied to this problem also required significant manual annotation. Results We describe Transposon Annotation Poisson Distribution Association Network Connectivity Environment (TAPDANCE software, which automates the identification of CISs within transposon junction fragment insertion data. Starting with barcoded sequence data, the software identifies and trims sequences and maps putative genomic sequence to a reference genome using the bowtie short read mapper. Poisson distribution statistics are then applied to assess and rank genomic regions showing significant enrichment for transposon insertion. Novel methods of counting insertions are used to ensure that the results presented have the expected characteristics of informative CISs. A persistent mySQL database is generated and utilized to keep track of sequences, mappings and common insertion sites. Additionally, associations between phenotypes and CISs are also identified using Fisher’s exact test with multiple testing correction. In a case study using previously published data we show that the TAPDANCE software identifies CISs as previously described, prioritizes them based on p-value, allows holistic visualization of the data within genome browser software and identifies relationships present in the structure of the data. Conclusions The TAPDANCE process is fully automated, performs similarly to previous labor intensive approaches

  1. Multiple homoplasious insertions and deletions of a Triticeae (Poaceae DNA transposon: a phylogenetic perspective

    Directory of Open Access Journals (Sweden)

    Mason-Gamer Roberta J

    2007-06-01

    Full Text Available Abstract Background Stowaway elements are short, non-autonomous DNA transposons categorized as miniature inverted-repeat transposable elements (MITEs. The high MITE copy number in grass genomes suggests an active history of amplification and insertion, but ongoing MITE activity has only rarely been seen, and ongoing Stowaway activity has never been observed. Thus, a phylogenetic perspective on presence vs. absence of elements in an aligned data set can provide valuable historical insights into the dynamics of MITE acquisition and loss. Results A Stowaway-like element resides within the fourth intron of a β-amylase gene in representatives of five genera in the wheat tribe, Triticeae. Its presence vs. absence was examined with reference to the β-amylase gene tree topology, and in light of sequence comparisons of the β-amylase elements to Triticeae Stowaway elements in the Entrez nucleotide database. Among the sequences lacking the element, there are five distinct putative excision footprints (one widespread and four restricted to unrelated lineages and two flanking deletions. The sequences that do contain elements are polyphyletic on the β-amylase tree, and their elements are divergent at the sequence level. The β-amylase elements do not form a monophyletic group relative to other Stowaway elements in Entrez; most are more similar to elements from other loci in other Triticeae genomes than they are to one another. Conclusion Combined, the phylogenetic distribution, sequence variation, and Entrez database comparisons indicate that a Stowaway-like element has undergone multiple deletions from and insertions into the same site in β-amylase intron 4 during the history of the tribe. The elements currently at the site represent multiple, distinct lineages that transcend generic boundaries. While patterns of Stowaway polymorphism across a phylogenetic data set do not allow evolutionary mechanisms to be inferred with certainty, they do provide

  2. TcA, the putative transposase of the C. elegans Tc1 transposon, has an N-terminal DNA binding domain.

    OpenAIRE

    Schukkink, R F; Plasterk, R H

    1990-01-01

    Tc1 is a transposon present in several copies in the genome of all natural isolates of the nematode C.elegans; it is actively transposing in many strains. In those strains Tc1 insertion is the main cause of spontaneous mutations. The transposon contains one large ORF that we call TcA; we assume that the TcA protein is the transposase of Tc1. We expressed TcA in E.coli, purified the protein and showed that it has a strong affinity for DNA (both single stranded and double stranded). A fusion pr...

  3. Transposon Invasion of the Paramecium Germline Genome Countered by a Domesticated PiggyBac Transposase and the NHEJ Pathway

    Science.gov (United States)

    Dubois, Emeline; Bischerour, Julien; Marmignon, Antoine; Mathy, Nathalie; Régnier, Vinciane; Bétermier, Mireille

    2012-01-01

    Sequences related to transposons constitute a large fraction of extant genomes, but insertions within coding sequences have generally not been tolerated during evolution. Thanks to their unique nuclear dimorphism and to their original mechanism of programmed DNA elimination from their somatic nucleus (macronucleus), ciliates are emerging model organisms for the study of the impact of transposable elements on genomes. The germline genome of the ciliate Paramecium, located in its micronucleus, contains thousands of short intervening sequences, the IESs, which interrupt 47% of genes. Recent data provided support to the hypothesis that an evolutionary link exists between Paramecium IESs and Tc1/mariner transposons. During development of the macronucleus, IESs are excised precisely thanks to the coordinated action of PiggyMac, a domesticated piggyBac transposase, and of the NHEJ double-strand break repair pathway. A PiggyMac homolog is also required for developmentally programmed DNA elimination in another ciliate, Tetrahymena. Here, we present an overview of the life cycle of these unicellular eukaryotes and of the developmentally programmed genome rearrangements that take place at each sexual cycle. We discuss how ancient domestication of a piggyBac transposase might have allowed Tc1/mariner elements to spread throughout the germline genome of Paramecium, without strong counterselection against insertion within genes. PMID:22888464

  4. Transposon-mediated random gene disruption with moderate halophilic bacteria and its application for halophilic bacterial siderophore analysis.

    Science.gov (United States)

    Matsui, Toru; Nishino, Tomohiko

    2016-12-01

    Analytical conditions using chromo azurol S was validated for quantification of siderophore in aqueous samples, followed by the characterization of siderophore derived from newly isolated moderately halophilic bacteria. Conditions with good linearity between the absorbance and the siderophore concentration were obtained at a siderophore concentration less than 20 µM, in the wavelength range between 630 and 660 nm with developing time for at least 2 h. Of the halophilic bacteria isolated from Tunisian soil, Halomonas sp., namely strain 21a was selected as siderophore producing halophiles. The strain produced siderophore significantly in the absence of iron in minimal medium. Siderophore-deficient mutant, namely IIa10, of the strain 21a was obtained from gene disruptant library constructed using transposon complex by electroporation. Genomic sequence analysis of the mutant IIa10 revealed that the transposon-inserted gene was TonB-dependent receptor. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Transgenerational changes in plant physiology and in transposon expression in response to UV-C stress in Arabidopsis thaliana.

    Science.gov (United States)

    Migicovsky, Zoe; Kovalchuk, Igor

    2014-01-01

    Stress has a negative impact on crop yield by altering a gain in biomass and affecting seed set. Recent reports suggest that exposure to stress also influences the response of the progeny. In this paper, we analyzed seed size, leaf size, bolting time and transposon expression in 2 consecutive generations of Arabidopsis thaliana plants exposed to moderate UV-C stress. Since previous reports suggested a potential role of Dicer-like (DCL) proteins in the establishment of transgenerational response, we used dcl2, dcl3 and dcl4 mutants in parallel with wild-type plants. These studies revealed that leaf number decreased in the progeny of UV-C stressed plants, and bolting occurred later. Transposons were also re-activated in the progeny of stressed plants. Changes in the dcl mutants were less prominent than in wild-type plants. DCL2 and DCL3 appeared to be more important in the transgenerational stress memory than DCL4 because transgenerational changes were less profound in the dcl2 and dcl3 mutants.

  6. R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata

    Energy Technology Data Exchange (ETDEWEB)

    Youvan, D C [Univ. of California, Berkeley; Elder, J T; Sandlin, D E; Zsebo, K; Alder, D P; Panopoulos, N J; Marrs, B L; Hearst, J E

    1982-01-01

    Site-directed mutagenesis of the photosynthetic apparatus (PSA) genes in Rhodopseudomonas capsulata is presented utilizing a transposon Tn7 mutagenized R-prime. The R-prime, pRPS404, bears most of the genes necessary for the differentiation of the photosynthetic apparatus. Mutagenesis of the R-prime with Tn7 in Escherichia coli, conjugation into R. capsulata, and homologous recombination with the wild-type alleles efficiently generates photosynthetic apparatus lesions. Wild-type alleles are lost spontaneously and the Tn7-induced lesions are revealed by subsequent intramolecular recombination between IS21 insertion elements that bracket the prime sequences in direct repeat. The molecular nature of the intermediates involved in the transposition, recombination and deletion have been investigated by Southern hybridization analysis. The spontaneous loss of wild-type alleles after homologous recombination with the chromosome may be of general use to other prokaryotic site-directed transposon mutagenesis schemes. The IS21-mediated deletion of the prime DNA is dependent on the RecA protein in E. coli, generating the parental R-factor bearing one IS21 element. A genetic-physical map exists for a portion of the prime photosynthetic apparatus DNA. When Tn7 is inserted into a bacteriochlorophyll gene in the R-prime and then crossed into R. capsulata, mutants are produced that accumulate a bacteriochlorophyll precursor, which is in excellent agreement with the existing genetic-physical map. This corroborates the mutagenesis scheme.

  7. Two hAT transposon genes were transferred from Brassicaceae to broomrapes and are actively expressed in some recipients

    Science.gov (United States)

    Sun, Ting; Renner, Susanne S.; Xu, Yuxing; Qin, Yan; Wu, Jianqiang; Sun, Guiling

    2016-01-01

    A growing body of evidence is pointing to an important role of horizontal gene transfer (HGT) in the evolution of higher plants. However, reports of HGTs of transposable elements (TEs) in plants are still scarce, and only one case is known of a class II transposon horizontally transferred between grasses. To investigate possible TE transfers in dicots, we performed transcriptome screening in the obligate root parasite Phelipanche aegyptiaca (Orobanchaceae), data-mining in the draft genome assemblies of four other Orobanchaceae, gene cloning, gene annotation in species with genomic information, and a molecular phylogenetic analysis. We discovered that the broomrape genera Phelipanche and Orobanche acquired two related nuclear genes (christened BO transposase genes), a new group of the hAT superfamily of class II transposons, from Asian Sisymbrieae or a closely related tribe of Brassicaceae, by HGT. The collinearity of the flanking genes, lack of a classic border structure, and low expression levels suggest that BO transposase genes cannot transpose in Brassicaceae, whereas they are highly expressed in P. aegyptiaca. PMID:27452947

  8. Enhanced Expression of Anti-CD19 Chimeric Antigen Receptor in piggyBac Transposon-Engineered T Cells

    Directory of Open Access Journals (Sweden)

    Daisuke Morita

    2018-03-01

    Full Text Available Adoptive T cell therapy using chimeric antigen receptor (CAR-modified T cells is a promising cancer immunotherapy. We previously developed a non-viral method of gene transfer into T cells using a piggyBac transposon system to improve the cost-effectiveness of CAR-T cell therapy. Here, we have further improved our technology by a novel culture strategy to increase the transfection efficiency and to reduce the time of T cell manufacturing. Using a CH2CH3-free CD19-specific CAR transposon vector and combining irradiated activated T cells (ATCs as feeder cells and virus-specific T cell receptor (TCR stimulation, we achieved 51.4% ± 14% CAR+ T cells and 2.8-fold expansion after 14 culture days. Expanded CD19.CAR-T cells maintained a significant fraction of CD45RA+CCR7+ T cells and demonstrated potent antitumor activity against CD19+ leukemic cells both in vitro and in vivo. Therefore, piggyBac-based gene transfer may provide an alternative to viral gene transfer for CAR-T cell therapy.

  9. Xanthomonas oryzae pv oryzae the Causal Agent of Bacterial Leaf Blight of rice: Isolation, Characterization, and Study of Transposon Mutagenesis

    Directory of Open Access Journals (Sweden)

    Abdjad Asih Nawangsih

    2011-04-01

    Full Text Available Xanthomonas oryzae pv oryzae the Causal Agent of Bacterial Leaf Blight of rice: Isolation, Characterization, and Study of Transposon Mutagenesis. X. oryzae pv. oryzae (Xoo causes bacterial leaf blight (BLB of rice (Oryza sativa L., a major disease that constrains production of the staple crop in many countries of the world. Identification of X. oryzae pv. oryzae (Xoo was conducted based on the disease symptoms, pathogenicity, morphological, physiological, and genetic characteristics of bacterial cultures isolated from the infected plants. Fifty bacterial isolates predicted as Xoo have been successfully isolated. They are aerobic, rod shaped, and Gram negative bacteria. The isolates were evaluated for their hypersensitivity in tobacco and pathogenicity in rice plant. Fifty isolates induced hypersensitive reaction in tobacco and showed pathogenicity symptom in rice in different length. Based on physiological test, hypersensitivity and pathogenicity reactions, three bacterial isolates strongly predicted as Xoo, i.e. STG21, STG42, and STG46, were non indole formation, non pigment fluorescent, hydrolyzed casein, catalase activity positive, but negative oxidase. Partial sequencing of 16S rRNA genes of STG21 and STG42 showed 80% and 82% homology with X. oryzae, respectively, while STG46 showed 84% homology with X. campestris. Mini-Tn5 transposon mutagenesis of STG21 generated one of the mutants (M5 lossed it’s ability to induce hypersensitive reaction in tobacco plant and deficient in pathogenicity on rice. The lesion length of rice leaf caused by the mutant M5 decreased up to 80%.

  10. Genetic Transformation of Hordeum vulgare ssp. spontaneum for the Development of a Transposon-Based Insertional Mutagenesis System.

    Science.gov (United States)

    Cardinal, Marie-Josée; Kaur, Rajvinder; Singh, Jaswinder

    2016-10-01

    Domestication and intensive selective breeding of plants has triggered erosion of genetic diversity of important stress-related alleles. Researchers highlight the potential of using wild accessions as a gene source for improvement of cereals such as barley, which has major economic and social importance worldwide. Previously, we have successfully introduced the maize Ac/Ds transposon system for gene identification in cultivated barley. The objective of current research was to investigate the response of Hordeum vulgare ssp. spontaneum wild barley accessions in tissue culture to standardize parameters for introduction of Ac/Ds transposons through genetic transformation. We investigated the response of ten wild barley genotypes for callus induction, regenerative green callus induction and regeneration of fertile plants. The activity of exogenous Ac/Ds elements was observed through a transient assay on immature wild barley embryos/callus whereby transformed embryos/calli were identified by the expression of GUS. Transient Ds expression bombardment experiments were performed on 352 pieces of callus (3-5 mm each) or immature embryos in 4 genotypes of wild barley. The transformation frequency of putative transgenic callus lines based on transient GUS expression ranged between 72 and100 % in wild barley genotypes. This is the first report of a transformation system in H. vulgare ssp. spontaneum.

  11. Prolonged Expression of Secreted Enzymes in Dogs After Liver-Directed Delivery of Sleeping Beauty Transposons: Implications for Non-Viral Gene Therapy of Systemic Disease.

    Science.gov (United States)

    Aronovich, Elena L; Hyland, Kendra A; Hall, Bryan C; Bell, Jason B; Olson, Erik R; Rusten, Myra Urness; Hunter, David W; Ellinwood, N Matthew; McIvor, R Scott; Hackett, Perry B

    2017-07-01

    The non-viral, integrating Sleeping Beauty (SB) transposon system is efficient in treating systemic monogenic disease in mice, including hemophilia A and B caused by deficiency of blood clotting factors and mucopolysaccharidosis types I and VII caused by α-L-iduronidase (IDUA) and β-glucuronidase (GUSB) deficiency, respectively. Modified approaches of the hydrodynamics-based procedure to deliver transposons to the liver in dogs were recently reported. Using the transgenic canine reporter secreted alkaline phosphatase (cSEAP), transgenic protein in the plasma was demonstrated for up to 6 weeks post infusion. This study reports that immunosuppression of dogs with gadolinium chloride (GdCl 3 ) prolonged the presence of cSEAP in the circulation up to 5.5 months after a single vector infusion. Transgene expression declined gradually but appeared to stabilize after about 2 months at approximately fourfold baseline level. Durability of transgenic protein expression in the plasma was inversely associated with transient increase of liver enzymes alanine transaminase and aspartate transaminase in response to the plasmid delivery procedure, which suggests a deleterious effect of hepatocellular toxicity on transgene expression. GdCl 3 treatment was ineffective for repeat vector infusions. In parallel studies, dogs were infused with potentially therapeutic transposons. Activities of transgenic IDUA and GUSB in plasma peaked at 50-350% of wildtype, but in the absence of immunosuppression lasted only a few days. Transposition was detectable by excision assay only when the most efficient transposase, SB100X, was used. Dogs infused with transposons encoding canine clotting factor IX (cFIX) were treated with GdCl 3 and showed expression profiles similar to those in cSEAP-infused dogs, with expression peaking at 40% wt (2 μg/mL). It is concluded that GdCl 3 can support extended transgene expression after hydrodynamic introduction of SB transposons in dogs, but that alternative

  12. Drifter, a novel, low copy hAT-like transposon in Fusarium oxysporum is activated during starvation.

    Science.gov (United States)

    Rep, Martijn; van der Does, H Charlotte; Cornelissen, Ben J C

    2005-06-01

    The facultative pathogenic fungus Fusarium oxysporum is known to harbour many different transposable and/or repetitive elements. We have identified Drifter, a novel DNA transposon of the hAT family in F. oxysporum. It was found adjoining SIX1-H, a truncated homolog of the SIX1 avirulence gene in F. oxysporum f. sp. lycopersici. Absence of a target site duplication as well as the 5' part of SIX1-H suggests that transposition of Drifter into the ancestor of SIX1-H was followed by loss of a chromosomal segment through recombination between Drifters. F. oxysporum isolates belonging to various formae speciales harbour between 0 and 5 full-length copies of Drifter and/or one or more copies with an internal deletion. Transcription of Drifter is activated during starvation for carbon or nitrogen.

  13. Identification of Genes Involved in Biofilm Formation and Respiration via Mini-Himar Transposon Mutagenesis of Geobacter sulfurreducens▿ †

    Science.gov (United States)

    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

  14. An active ac/ds transposon system for activation tagging in tomato cultivar m82 using clonal propagation.

    Science.gov (United States)

    Carter, Jared D; Pereira, Andy; Dickerman, Allan W; Veilleux, Richard E

    2013-05-01

    Tomato (Solanum lycopersicum) is a model organism for Solanaceae in both molecular and agronomic research. This project utilized Agrobacterium tumefaciens transformation and the transposon-tagging construct Activator (Ac)/Dissociator (Ds)-ATag-Bar_gosGFP to produce activation-tagged and knockout mutants in the processing tomato cultivar M82. The construct carried hygromycin resistance (hyg), green fluorescent protein (GFP), and the transposase (TPase) of maize (Zea mays) Activator major transcript X054214.1 on the stable Ac element, along with a 35S enhancer tetramer and glufosinate herbicide resistance (BAR) on the mobile Ds-ATag element. An in vitro propagation strategy was used to produce a population of 25 T0 plants from a single transformed plant regenerated in tissue culture. A T1 population of 11,000 selfed and cv M82 backcrossed progeny was produced from the functional T0 line. This population was screened using glufosinate herbicide, hygromycin leaf painting, and multiplex polymerase chain reaction (PCR). Insertion sites of transposed Ds-ATag elements were identified through thermal asymmetric interlaced PCR, and resulting product sequences were aligned to the recently published tomato genome. A population of 509 independent, Ds-only transposant lines spanning all 12 tomato chromosomes has been developed. Insertion site analysis demonstrated that more than 80% of these lines harbored Ds insertions conducive to activation tagging. The capacity of the Ds-ATag element to alter transcription was verified by quantitative real-time reverse transcription-PCR in two mutant lines. The transposon-tagged lines have been immortalized in seed stocks and can be accessed through an online database, providing a unique resource for tomato breeding and analysis of gene function in the background of a commercial tomato cultivar.

  15. Role of transposon-derived small RNAs in the interplay between genomes and parasitic DNA in rice.

    Directory of Open Access Journals (Sweden)

    Misuzu Nosaka

    2012-09-01

    Full Text Available RNA silencing is a defense system against "genomic parasites" such as transposable elements (TE, which are potentially harmful to host genomes. In plants, transcripts from TEs induce production of double-stranded RNAs (dsRNAs and are processed into small RNAs (small interfering RNAs, siRNAs that suppress TEs by RNA-directed DNA methylation. Thus, the majority of TEs are epigenetically silenced. On the other hand, most of the eukaryotic genome is composed of TEs and their remnants, suggesting that TEs have evolved countermeasures against host-mediated silencing. Under some circumstances, TEs can become active and increase in copy number. Knowledge is accumulating on the mechanisms of TE silencing by the host; however, the mechanisms by which TEs counteract silencing are poorly understood. Here, we show that a class of TEs in rice produces a microRNA (miRNA to suppress host silencing. Members of the microRNA820 (miR820 gene family are located within CACTA DNA transposons in rice and target a de novo DNA methyltransferase gene, OsDRM2, one of the components of epigenetic silencing. We confirmed that miR820 negatively regulates the expression of OsDRM2. In addition, we found that expression levels of various TEs are increased quite sensitively in response to decreased OsDRM2 expression and DNA methylation at TE loci. Furthermore, we found that the nucleotide sequence of miR820 and its recognition site within the target gene in some Oryza species have co-evolved to maintain their base-pairing ability. The co-evolution of these sequences provides evidence for the functionality of this regulation. Our results demonstrate how parasitic elements in the genome escape the host's defense machinery. Furthermore, our analysis of the regulation of OsDRM2 by miR820 sheds light on the action of transposon-derived small RNAs, not only as a defense mechanism for host genomes but also as a regulator of interactions between hosts and their parasitic elements.

  16. High-Level Heat Resistance of Spores of Bacillus amyloliquefaciens and Bacillus licheniformis Results from the Presence of a spoVA Operon in a Tn1546 Transposon

    Science.gov (United States)

    Berendsen, Erwin M.; Koning, Rosella A.; Boekhorst, Jos; de Jong, Anne; Kuipers, Oscar P.; Wells-Bennik, Marjon H. J.

    2016-01-01

    Bacterial endospore formers can produce spores that are resistant to many food processing conditions, including heat. Some spores may survive heating processes aimed at production of commercially sterile foods. Recently, it was shown that a spoVA operon, designated spoVA2mob, present on a Tn1546 transposon in Bacillus subtilis, leads to profoundly increased wet heat resistance of B. subtilis spores. Such Tn1546 transposon elements including the spoVA2mob operon were also found in several strains of Bacillus amyloliquefaciens and Bacillus licheniformis, and these strains were shown to produce spores with significantly higher resistances to wet heat than their counterparts lacking this transposon. In this study, the locations and compositions of Tn1546 transposons encompassing the spoVA2mob operons in B. amyloliquefaciens and B. licheniformis were analyzed. Introduction of these spoVA2mob operons into B. subtilis 168 (producing spores that are not highly heat resistant) rendered mutant 168 strains that produced high-level heat resistant spores, demonstrating that these elements in B. amyloliquefaciens and B. licheniformis are responsible for high level heat resistance of spores. Assessment of growth of the nine strains of each species between 5.2°C and 57.7°C showed some differences between strains, especially at lower temperatures, but all strains were able to grow at 57.7°C. Strains of B. amyloliquefaciens and B. licheniformis that contain the Tn1546 elements (and produce high-level heat resistant spores) grew at temperatures similar to those of their Tn1546-negative counterparts that produce low-level heat resistant spores. The findings presented in this study allow for detection of B. amyloliquefaciens and B. licheniformis strains that produce highly heat resistant spores in the food chain. PMID:27994575

  17. Altered expression of testis-specific genes, piRNAs, and transposons in the silkworm ovary masculinized by a W chromosome mutation

    Science.gov (United States)

    2012-01-01

    Background In the silkworm, Bombyx mori, femaleness is strongly controlled by the female-specific W chromosome. Originally, it was presumed that the W chromosome encodes female-determining gene(s), accordingly called Fem. However, to date, neither Fem nor any protein-coding gene has been identified from the W chromosome. Instead, the W chromosome is occupied with numerous transposon-related sequences. Interestingly, the silkworm W chromosome is a source of female-enriched PIWI-interacting RNAs (piRNAs). piRNAs are small RNAs of 23-30 nucleotides in length, which are required for controlling transposon activity in animal gonads. A recent study has identified a novel mutant silkworm line called KG, whose mutation in the W chromosome causes severe female masculinization. However, the molecular nature of KG line has not been well characterized yet. Results Here we molecularly characterize the KG line. Genomic PCR analyses using currently available W chromosome-specific PCR markers indicated that no large deletion existed in the KG W chromosome. Genetic analyses demonstrated that sib-crosses within the KG line suppressed masculinization. Masculinization reactivated when crossing KG females with wild type males. Importantly, the KG ovaries exhibited a significantly abnormal transcriptome. First, the KG ovaries misexpressed testis-specific genes. Second, a set of female-enriched piRNAs was downregulated in the KG ovaries. Third, several transposons were overexpressed in the KG ovaries. Conclusions Collectively, the mutation in the KG W chromosome causes broadly altered expression of testis-specific genes, piRNAs, and transposons. To our knowledge, this is the first study that describes a W chromosome mutant with such an intriguing phenotype. PMID:22452797

  18. nanos-Driven expression of piggyBac transposase induces mobilization of a synthetic autonomous transposon in the malaria vector mosquito, Anopheles stephensi.

    Science.gov (United States)

    Macias, Vanessa M; Jimenez, Alyssa J; Burini-Kojin, Bianca; Pledger, David; Jasinskiene, Nijole; Phong, Celine Hien; Chu, Karen; Fazekas, Aniko; Martin, Kelcie; Marinotti, Osvaldo; James, Anthony A

    2017-08-01

    Transposons are a class of selfish DNA elements that can mobilize within a genome. If mobilization is accompanied by an increase in copy number (replicative transposition), the transposon may sweep through a population until it is fixed in all of its interbreeding members. This introgression has been proposed as the basis for drive systems to move genes with desirable phenotypes into target species. One such application would be to use them to move a gene conferring resistance to malaria parasites throughout a population of vector mosquitos. We assessed the feasibility of using the piggyBac transposon as a gene-drive mechanism to distribute anti-malarial transgenes in populations of the malaria vector, Anopheles stephensi. We designed synthetic gene constructs that express the piggyBac transposase in the female germline using the control DNA of the An. stephensi nanos orthologous gene linked to marker genes to monitor inheritance. Two remobilization events were observed with a frequency of one every 23 generations, a rate far below what would be useful to drive anti-pathogen transgenes into wild mosquito populations. We discuss the possibility of optimizing this system and the impetus to do so. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. Genome-Wide Transposon Mutagenesis Indicates that Mycobacterium marinum Customizes Its Virulence Mechanisms for Survival and Replication in Different Hosts

    KAUST Repository

    Weerdenburg, Eveline M.

    2015-02-17

    The interaction of environmental bacteria with unicellular eukaryotes is generally considered a major driving force for the evolution of intracellular pathogens, allowing them to survive and replicate in phagocytic cells of vertebrate hosts. To test this hypothesis on a genome-wide level, we determined for the intracellular pathogen Mycobacterium marinum whether it uses conserved strategies to exploit host cells from both protozoan and vertebrate origin. Using transposon-directed insertion site sequencing (TraDIS), we determined differences in genetic requirements for survival and replication in phagocytic cells of organisms from different kingdoms. In line with the general hypothesis, we identified a number of general virulence mechanisms, including the type VII protein secretion system ESX-1, biosynthesis of polyketide lipids, and utilization of sterols. However, we were also able to show that M. marinum contains an even larger set of host-specific virulence determinants, including proteins involved in the modification of surface glycolipids and, surprisingly, the auxiliary proteins of the ESX-1 system. Several of these factors were in fact counterproductive in other hosts. Therefore, M. marinum contains different sets of virulence factors that are tailored for specific hosts. Our data imply that although amoebae could function as a training ground for intracellular pathogens, they do not fully prepare pathogens for crossing species barriers.

  20. Long-Term PEDF Release in Rat Iris and Retinal Epithelial Cells after Sleeping Beauty Transposon-Mediated Gene Delivery

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    Laura Garcia-Garcia

    2017-12-01

    Full Text Available Pigment epithelium derived factor (PEDF is a potent antiangiogenic, neurotrophic, and neuroprotective molecule that is the endogenous inhibitor of vascular endothelial growth factor (VEGF in the retina. An ex vivo gene therapy approach based on transgenic overexpression of PEDF in the eye is assumed to rebalance the angiogenic-antiangiogenic milieu of the retina, resulting in growth regression of choroidal blood vessels, the hallmark of neovascular age-related macular degeneration. Here, we show that rat pigment epithelial cells can be efficiently transfected with the PEDF-expressing non-viral hyperactive Sleeping Beauty transposon system delivered in a form free of antibiotic resistance marker miniplasmids. The engineered retinal and iris pigment epithelium cells secrete high (141 ± 13 and 222 ± 14 ng PEDF levels in 72 hr in vitro. In vivo studies showed cell survival and insert expression during at least 4 months. Transplantation of the engineered cells to the subretinal space of a rat model of choroidal neovascularization reduces almost 50% of the development of new vessels.

  1. Generation of comprehensive transposon insertion mutant library for the model archaeon, Haloferax volcanii, and its use for gene discovery.

    Science.gov (United States)

    Kiljunen, Saija; Pajunen, Maria I; Dilks, Kieran; Storf, Stefanie; Pohlschroder, Mechthild; Savilahti, Harri

    2014-12-09

    Archaea share fundamental properties with bacteria and eukaryotes. Yet, they also possess unique attributes, which largely remain poorly characterized. Haloferax volcanii is an aerobic, moderately halophilic archaeon that can be grown in defined media. It serves as an excellent archaeal model organism to study the molecular mechanisms of biological processes and cellular responses to changes in the environment. Studies on haloarchaea have been impeded by the lack of efficient genetic screens that would facilitate the identification of protein functions and respective metabolic pathways. Here, we devised an insertion mutagenesis strategy that combined Mu in vitro DNA transposition and homologous-recombination-based gene targeting in H. volcanii. We generated an insertion mutant library, in which the clones contained a single genomic insertion. From the library, we isolated pigmentation-defective and auxotrophic mutants, and the respective insertions pinpointed a number of genes previously known to be involved in carotenoid and amino acid biosynthesis pathways, thus validating the performance of the methodologies used. We also identified mutants that had a transposon insertion in a gene encoding a protein of unknown or putative function, demonstrating that novel roles for non-annotated genes could be assigned. We have generated, for the first time, a random genomic insertion mutant library for a halophilic archaeon and used it for efficient gene discovery. The library will facilitate the identification of non-essential genes behind any specific biochemical pathway. It represents a significant step towards achieving a more complete understanding of the unique characteristics of halophilic archaea.

  2. Molecular characterization and diversity of a novel non-autonomous mutator-like transposon family in brassica

    International Nuclear Information System (INIS)

    Nouroz, F.

    2015-01-01

    Transposable elements (TEs) are capable of mobilizing from one genomic location to other, with changes in their copy numbers. Mutator-like elements (MULEs) are DNA transposons characterized by 9 bp target site duplications (TSDs), with high variability in sequence and length, and include non-conserved terminal inverted repeats (TIRs). We identified and characterized a family of Mutator-like elements designated as Shahroz. The structural and molecular analyses revealed that family had a small number of mostly defective non-autonomous MULEs and has shown limited activity in the evolutionary history of the Brassica A-genome. The Shahroz elements range in size from 2734 to 3160 bp including 76 bp imperfect TIRs and 9 bp variable TSDs. The individual copies have shown high homology (52-99%) in their entire lengths. The study revealed that the elements are less in numbers but active in Brassica rapa genomes and PCR amplification revealed their specificity and amplification in A-genome containing diploid and polyploids Brassica. The phylogenetic analysis of Brassica MULEs with other plant Mutator elements revealed that no correlation exists between Brassica MULEs and other elements suggesting a separate line of evolution. Analyzing the regions flanking the insertions revealed that the insertions have showed a preference for AT rich regions. The detailed study of these insertions revealed that although less in number and small sizes, they have played a role in Brassica genome evolution by their mobilization. (author)

  3. The Identification of Genes Important in Pseudomonas syringae pv. phaseolicola Plant Colonisation Using In Vitro Screening of Transposon Libraries.

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    Bharani Manoharan

    Full Text Available The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms around plant cells. If the pathogen can suppress the plant's natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction.

  4. Development of stable reporter system cloning luxCDABE genes into chromosome of Salmonella enterica serotypes using Tn7 transposon

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    Lawrence Mark L

    2010-07-01

    Full Text Available Abstract Background Salmonellosis may be a food safety problem when raw food products are mishandled and not fully cooked. In previous work, we developed bioluminescent Salmonella enterica serotypes using a plasmid-based reporting system that can be used for real-time monitoring of the pathogen's growth on food products in short term studies. In this study, we report the use of a Tn7-based transposon system for subcloning of luxCDABE genes into the chromosome of eleven Salmonella enterica serotypes isolated from the broiler production continuum. Results We found that the lux operon is constitutively expressed from the chromosome post-transposition and the lux cassette is stable without external pressure, i.e. antibiotic selection, for all Salmonella enterica serotypes used. Bioluminescence expression is based on an active electron transport chain and is directly related with metabolic activity. This relationship was quantified by measuring bioluminescence against a temperature gradient in aqueous solution using a luminometer. In addition, bioluminescent monitoring of two serotypes confirmed that our chicken skin model has the potential to be used to evaluate pathogen mitigation strategies. Conclusions This study demonstrated that our new stable reporting system eliminates bioluminescence variation due to plasmid instability and provides a reliable real-time experimental system to study application of preventive measures for Salmonella on food products in real-time for both short and long term studies.

  5. Autoantigen La promotes efficient RNAi, antiviral response, and transposon silencing by facilitating multiple-turnover RISC catalysis.

    Science.gov (United States)

    Liu, Ying; Tan, Huiling; Tian, Hui; Liang, Chunyang; Chen, She; Liu, Qinghua

    2011-11-04

    The effector of RNA interference (RNAi) is the RNA-induced silencing complex (RISC). C3PO promotes the activation of RISC by degrading the Argonaute2 (Ago2)-nicked passenger strand of duplex siRNA. Active RISC is a multiple-turnover enzyme that uses the guide strand of siRNA to direct the Ago2-mediated sequence-specific cleavage of complementary mRNA. How this effector step of RNAi is regulated is currently unknown. Here, we used the human Ago2 minimal RISC system to purify Sjögren's syndrome antigen B (SSB)/autoantigen La as an activator of the RISC-mediated mRNA cleavage activity. Our reconstitution studies showed that La could promote multiple-turnover RISC catalysis by facilitating the release of cleaved mRNA from RISC. Moreover, we demonstrated that La was required for efficient RNAi, antiviral defense, and transposon silencing in vivo. Taken together, the findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. A TALE of transposition: Tn3-like transposons play a major role in the spread of pathogenicity determinants of Xanthomonas citri and other xanthomonads.

    Science.gov (United States)

    Ferreira, Rafael Marini; de Oliveira, Amanda Carolina P; Moreira, Leandro M; Belasque, José; Gourbeyre, Edith; Siguier, Patricia; Ferro, Maria Inês T; Ferro, Jesus A; Chandler, Michael; Varani, Alessandro M

    2015-02-17

    Members of the genus Xanthomonas are among the most important phytopathogens. A key feature of Xanthomonas pathogenesis is the translocation of type III secretion system (T3SS) effector proteins (T3SEs) into the plant target cells via a T3SS. Several T3SEs and a murein lytic transglycosylase gene (mlt, required for citrus canker symptoms) are found associated with three transposition-related genes in Xanthomonas citri plasmid pXAC64. These are flanked by short inverted repeats (IRs). The region was identified as a transposon, TnXax1, with typical Tn3 family features, including a transposase and two recombination genes. Two 14-bp palindromic sequences within a 193-bp potential resolution site occur between the recombination genes. Additional derivatives carrying different T3SEs and other passenger genes occur in different Xanthomonas species. The T3SEs include transcription activator-like effectors (TALEs). Certain TALEs are flanked by the same IRs as found in TnXax1 to form mobile insertion cassettes (MICs), suggesting that they may be transmitted horizontally. A significant number of MICs carrying other passenger genes (including a number of TALE genes) were also identified, flanked by the same TnXax1 IRs and delimited by 5-bp target site duplications. We conclude that a large fraction of T3SEs, including individual TALEs and potential pathogenicity determinants, have spread by transposition and that TnXax1, which exhibits all of the essential characteristics of a functional transposon, may be involved in driving MIC transposition. We also propose that TALE genes may diversify by fork slippage during the replicative Tn3 family transposition. These mechanisms may play a crucial role in the emergence of Xanthomonas pathogenicity. Xanthomonas genomes carry many insertion sequences (IS) and transposons, which play an important role in their evolution and architecture. This study reveals a key relationship between transposons and pathogenicity determinants in

  7. Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway

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    Gaigalat Lars

    2006-08-01

    Full Text Available Abstract Background Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. Results A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100-containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4-monophosphatases (EC 3.1.3.25. Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown

  8. Putative adaptive inter-slope divergence of transposon frequency in fruit flies (Drosophila melanogaster) at "Evolution Canyon", Mount Carmel, Israel.

    Science.gov (United States)

    Beiles, Avigdor; Raz, Shmuel; Ben-Abu, Yuval; Nevo, Eviatar

    2015-10-14

    The current analysis of transposon elements (TE) in Drosophila melanogaster at Evolution Canyon, (EC), Israel, is based on data and analysis done by our collaborators (Drs. J. Gonzalez, J. Martinez and W. Makalowski, this issue). They estimated the frequencies of 28 TEs (transposon elements) in fruit flies (D. melanogaster) from the ecologically tropic, hot, and dry south-facing slope (SFS) or "African" slope (AS) of EC and compared it with the TE frequencies on the temperate-cool and humid north-facing slope (NFS) or "European" slope (ES), separated, on average, by 250 m. The flies were sampled from two stations on each slope. We received their results, including the frequencies of each TE on each slope, and the probabilities of the statistical analyses (G-tests) of each TE separately. We continued the analysis of the inter-slope differences of the frequencies of the TEs, and based our different conclusions on that analysis and on the difference between micro (=EC) and macro (2000 km.) comparisons [Gonzalez et al. 2015 doi: 10.1186/s13062-015-0075-4 ]. Our collaborators based all their conclusions on the non-significant results of each of the individual tests of the 28 TEs. We analysed also the distribution of the TE differences between the slopes, based on their results. Thirteen TEs were more frequent on the SFS, 11 were more frequent on the NFS, and four had equal frequencies. Because of the equalizing effect of the ongoing migration, only small and temporary differences between the slopes (0 - 0.06) were regarded by us as random fluctuations (drift). Three TEs were intermediate (0.08-0.09) and await additional research. The 11 TEs with large frequency differences (0.12 - 0.22) were regarded by us as putative adaptive TEs, because the equalizing power of ongoing migration will eliminate random large differences. Five of them were higher on the SFS and six were higher on the NFS. Gaps in the distribution of the differences distinguished between the large and

  9. Striking structural dynamism and nucleotide sequence variation of the transposon Galileo in the genome of Drosophila mojavensis.

    Science.gov (United States)

    Marzo, Mar; Bello, Xabier; Puig, Marta; Maside, Xulio; Ruiz, Alfredo

    2013-02-04

    Galileo is a transposable element responsible for the generation of three chromosomal inversions in natural populations of Drosophila buzzatii. Although the most characteristic feature of Galileo is the long internally-repetitive terminal inverted repeats (TIRs), which resemble the Drosophila Foldback element, its transposase-coding sequence has led to its classification as a member of the P-element superfamily (Class II, subclass 1, TIR order). Furthermore, Galileo has a wide distribution in the genus Drosophila, since it has been found in 6 of the 12 Drosophila sequenced genomes. Among these species, D. mojavensis, the one closest to D. buzzatii, presented the highest diversity in sequence and structure of Galileo elements. In the present work, we carried out a thorough search and annotation of all the Galileo copies present in the D. mojavensis sequenced genome. In our set of 170 Galileo copies we have detected 5 Galileo subfamilies (C, D, E, F, and X) with different structures ranging from nearly complete, to only 2 TIR or solo TIR copies. Finally, we have explored the structural and length variation of the Galileo copies that point out the relatively frequent rearrangements within and between Galileo elements. Different mechanisms responsible for these rearrangements are discussed. Although Galileo is a transposable element with an ancient history in the D. mojavensis genome, our data indicate a recent transpositional activity. Furthermore, the dynamism in sequence and structure, mainly affecting the TIRs, suggests an active exchange of sequences among the copies. This exchange could lead to new subfamilies of the transposon, which could be crucial for the long-term survival of the element in the genome.

  10. Insertion of transposon in the vicinity of SSK2 confers enhanced tolerance to furfural in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyun-Soo [Ewha Womans Univ., Seoul (Korea, Republic of). Dept. of Life Science; Kim, Na-Rae [Ewha Womans Univ., Seoul (Korea, Republic of). Div. of Life and Pharmaceutical Sciences; Kim, Wankee [Ewha Womans Univ., Seoul (Korea, Republic of). Dept. of Life Science; Ajou Univ., Suwon (Korea, Republic of). Inst. for Medical Sciences; Choi, Wonja [Ewha Womans Univ., Seoul (Korea, Republic of). Dept. of Life Science; Ewha Womans Univ., Seoul (Korea, Republic of). Microbial Resources Research Center

    2012-07-15

    Furfural is one of the major inhibitors generated during sugar production from cellulosic materials and, as an aldehyde, inhibits various cellular activities of microorganisms used, leading to prolonged lag time during ethanologenic fermentation. Since Saccharomyces cerevisiae strains tolerant to furfural are of great economic benefit in producing bioethanol, much effort to obtain more efficient strains continues to be made. In this study, we examined the furfural tolerance of transposon mutant strains (Tn 1-5) with enhanced ethanol tolerance and found that one of them (Tn 2), in which SSK2 is downregulated at the transcriptional level, displayed improved furfural tolerance. Such phenotype was abolished by complementation of the entire open reading frame of SSK2, which encodes a mitogen-activated protein (MAP) kinase kinase kinase of the high osmolarity glycerol (HOG) signaling pathway, suggesting an inhibitory effect of SSK2 in coping with furfural stress. Tn 2 showed a significant decrease in the intracellular level of reactive oxygen species (ROS) and early and high activation of Hog1p, a MAP kinase integral to the HOG pathway in response to furfural. The transcriptional levels of CTT1 and GLR1, two of known Hog1p downstream target genes whose protein products are involved in reducing ROS, were increased by 43 % and 56 % respectively compared with a control strain, probably resulting in the ROS decrease. Tn 2 also showed a shortened lag time during fermentation in the presence of furfural, resulting from efficient conversion of furfural to non-toxic (or less toxic) furfuryl alcohol. Taken together, the enhanced furfural tolerance of Tn 2 is suggested to be conferred by the combined effect of an early event of less ROS accumulation and a late event of efficient detoxification of furfural. (orig.)

  11. Insertion of transposon in the vicinity of SSK2 confers enhanced tolerance to furfural in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kim, Hyun-Soo; Kim, Na-Rae; Kim, Wankee; Choi, Wonja

    2012-07-01

    Furfural is one of the major inhibitors generated during sugar production from cellulosic materials and, as an aldehyde, inhibits various cellular activities of microorganisms used, leading to prolonged lag time during ethanologenic fermentation. Since Saccharomyces cerevisiae strains tolerant to furfural are of great economic benefit in producing bioethanol, much effort to obtain more efficient strains continues to be made. In this study, we examined the furfural tolerance of transposon mutant strains (Tn 1-5) with enhanced ethanol tolerance and found that one of them (Tn 2), in which SSK2 is downregulated at the transcriptional level, displayed improved furfural tolerance. Such phenotype was abolished by complementation of the entire open reading frame of SSK2, which encodes a mitogen-activated protein (MAP) kinase kinase kinase of the high osmolarity glycerol (HOG) signaling pathway, suggesting an inhibitory effect of SSK2 in coping with furfural stress. Tn 2 showed a significant decrease in the intracellular level of reactive oxygen species (ROS) and early and high activation of Hog1p, a MAP kinase integral to the HOG pathway in response to furfural. The transcriptional levels of CTT1 and GLR1, two of known Hog1p downstream target genes whose protein products are involved in reducing ROS, were increased by 43 % and 56 % respectively compared with a control strain, probably resulting in the ROS decrease. Tn 2 also showed a shortened lag time during fermentation in the presence of furfural, resulting from efficient conversion of furfural to non-toxic (or less toxic) furfuryl alcohol. Taken together, the enhanced furfural tolerance of Tn 2 is suggested to be conferred by the combined effect of an early event of less ROS accumulation and a late event of efficient detoxification of furfural.

  12. Identification and Characterization of Non-Cellulose-Producing Mutants of Gluconacetobacter hansenii Generated by Tn5 Transposon Mutagenesis

    Science.gov (United States)

    Deng, Ying; Nagachar, Nivedita; Xiao, Chaowen; Tien, Ming

    2013-01-01

    The acs operon of Gluconacetobacter is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel−) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript “Ax” indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel− mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel− mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding β-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis. PMID:24013627

  13. Early embryogenesis-specific expression of the rice transposon Ping enhances amplification of the MITE mPing.

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    Shota Teramoto

    2014-06-01

    Full Text Available Miniature inverted-repeat transposable elements (MITEs are numerically predominant transposable elements in the rice genome, and their activities have influenced the evolution of genes. Very little is known about how MITEs can rapidly amplify to thousands in the genome. The rice MITE mPing is quiescent in most cultivars under natural growth conditions, although it is activated by various stresses, such as tissue culture, gamma-ray irradiation, and high hydrostatic pressure. Exceptionally in the temperate japonica rice strain EG4 (cultivar Gimbozu, mPing has reached over 1000 copies in the genome, and is amplifying owing to its active transposition even under natural growth conditions. Being the only active MITE, mPing in EG4 is an appropriate material to study how MITEs amplify in the genome. Here, we provide important findings regarding the transposition and amplification of mPing in EG4. Transposon display of mPing using various tissues of a single EG4 plant revealed that most de novo mPing insertions arise in embryogenesis during the period from 3 to 5 days after pollination (DAP, and a large majority of these insertions are transmissible to the next generation. Locus-specific PCR showed that mPing excisions and insertions arose at the same time (3 to 5 DAP. Moreover, expression analysis and in situ hybridization analysis revealed that Ping, an autonomous partner for mPing, was markedly up-regulated in the 3 DAP embryo of EG4, whereas such up-regulation of Ping was not observed in the mPing-inactive cultivar Nipponbare. These results demonstrate that the early embryogenesis-specific expression of Ping is responsible for the successful amplification of mPing in EG4. This study helps not only to elucidate the whole mechanism of mPing amplification but also to further understand the contribution of MITEs to genome evolution.

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

    Science.gov (United States)

    Watabe, Kazuyuki; Mimuro, Mamoru; Tsuchiya, Tohru

    2014-11-01

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

  15. Tumor-directed gene therapy in mice using a composite nonviral gene delivery system consisting of the piggyBac transposon and polyethylenimine

    International Nuclear Information System (INIS)

    Kang, Yu; Zhang, Xiaoyan; Jiang, Wei; Wu, Chaoqun; Chen, Chunmei; Zheng, Yufang; Gu, Jianren; Xu, Congjian

    2009-01-01

    Compared with viral vectors, nonviral vectors are less immunogenic, more stable, safer and easier to replication for application in cancer gene therapy. However, nonviral gene delivery system has not been extensively used because of the low transfection efficiency and the short transgene expression, especially in vivo. It is desirable to develop a nonviral gene delivery system that can support stable genomic integration and persistent gene expression in vivo. Here, we used a composite nonviral gene delivery system consisting of the piggyBac (PB) transposon and polyethylenimine (PEI) for long-term transgene expression in mouse ovarian tumors. A recombinant plasmid PB [Act-RFP, HSV-tk] encoding both the herpes simplex thymidine kinase (HSV-tk) and the monomeric red fluorescent protein (mRFP1) under PB transposon elements was constructed. This plasmid and the PBase plasmid were injected into ovarian cancer tumor xenografts in mice by in vivo PEI system. The antitumor effects of HSV-tk/ganciclovir (GCV) system were observed after intraperitoneal injection of GCV. Histological analysis and TUNEL assay were performed on the cryostat sections of the tumor tissue. Plasmid construction was confirmed by PCR analysis combined with restrictive enzyme digestion. mRFP1 expression could be visualized three weeks after the last transfection of pPB/TK under fluorescence microscopy. After GCV admission, the tumor volume of PB/TK group was significantly reduced and the tumor inhibitory rate was 81.96% contrasted against the 43.07% in the TK group. Histological analysis showed that there were extensive necrosis and lymphocytes infiltration in the tumor tissue of the PB/TK group but limited in the tissue of control group. TUNEL assays suggested that the transfected cells were undergoing apoptosis after GCV admission in vivo. Our results show that the nonviral gene delivery system coupling PB transposon with PEI can be used as an efficient tool for gene therapy in ovarian cancer

  16. Genome-wide transposon mutagenesis of Proteus mirabilis: Essential genes, fitness factors for catheter-associated urinary tract infection, and the impact of polymicrobial infection on fitness requirements

    Science.gov (United States)

    Smith, Sara N.; Zhao, Lili; Wu, Weisheng

    2017-01-01

    The Gram-negative bacterium Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Numerous prior studies have uncovered virulence factors for P. mirabilis pathogenicity in a murine model of ascending UTI, but little is known concerning pathogenesis during CAUTI or polymicrobial infection. In this study, we utilized five pools of 10,000 transposon mutants each and transposon insertion-site sequencing (Tn-Seq) to identify the full arsenal of P. mirabilis HI4320 fitness factors for single-species versus polymicrobial CAUTI with Providencia stuartii BE2467. 436 genes in the input pools lacked transposon insertions and were therefore concluded to be essential for P. mirabilis growth in rich medium. 629 genes were identified as P. mirabilis fitness factors during single-species CAUTI. Tn-Seq from coinfection with P. stuartii revealed 217/629 (35%) of the same genes as identified by single-species Tn-Seq, and 1353 additional factors that specifically contribute to colonization during coinfection. Mutants were constructed in eight genes of interest to validate the initial screen: 7/8 (88%) mutants exhibited the expected phenotypes for single-species CAUTI, and 3/3 (100%) validated the expected phenotypes for polymicrobial CAUTI. This approach provided validation of numerous previously described P. mirabilis fitness determinants from an ascending model of UTI, the discovery of novel fitness determinants specifically for CAUTI, and a stringent assessment of how polymicrobial infection influences fitness requirements. For instance, we describe a requirement for branched-chain amino acid biosynthesis by P. mirabilis during coinfection due to high-affinity import of leucine by P. stuartii. Further investigation of genes and pathways that provide a competitive advantage during both single-species and polymicrobial CAUTI will likely provide robust targets for therapeutic intervention to reduce P. mirabilis

  17. Transposon defense by endo-siRNAs, piRNAs and somatic pilRNAs in Drosophila: contributions of Loqs-PD and R2D2.

    Directory of Open Access Journals (Sweden)

    Milijana Mirkovic-Hösle

    Full Text Available Transposable elements are a serious threat for genome integrity and their control via small RNA mediated silencing pathways is an ancient strategy. The fruit fly Drosophila melanogaster has two silencing activities that target transposons: endogenous siRNAs (esiRNAs or endo-siRNAs and Piwi-interacting small RNAs (piRNAs. The biogenesis of endo-siRNAs involves the Dicer-2 co-factors Loqs-PD, which acts predominantly during processing of dsRNA by Dcr-2, and R2D2, which primarily helps to direct siRNAs into the RNA interference effector Ago2. Nonetheless, loss of either protein is not sufficient to produce a phenotype comparable with a dcr-2 mutation. We provide further deep sequencing evidence supporting the notion that R2D2 and Loqs-PD have partially overlapping function. Certain transposons display a preference for either dsRBD-protein during production or loading; this appeared to correlate neither with overall abundance, classification of the transposon or a specific site of genomic origin. The endo-siRNA biogenesis pathway in germline operates according to the same principles as the existing model for the soma, and its impairment does not significantly affect piRNAs. Expanding the analysis, we confirmed the occurrence of somatic piRNA-like RNAs (pilRNAs that show a ping-pong signature. We detected expression of the Piwi-family protein mRNAs only barely above background, indicating that the somatic pilRNAs may arise from a small sub-population of somatic cells that express a functional piRNA pathway.

  18. The SUVR4 histone lysine methyltransferase binds ubiquitin and converts H3K9me1 to H3K9me3 on transposon chromatin in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Silje V Veiseth

    2011-03-01

    Full Text Available Chromatin structure and gene expression are regulated by posttranslational modifications (PTMs on the N-terminal tails of histones. Mono-, di-, or trimethylation of lysine residues by histone lysine methyltransferases (HKMTases can have activating or repressive functions depending on the position and context of the modified lysine. In Arabidopsis, trimethylation of lysine 9 on histone H3 (H3K9me3 is mainly associated with euchromatin and transcribed genes, although low levels of this mark are also detected at transposons and repeat sequences. Besides the evolutionarily conserved SET domain which is responsible for enzyme activity, most HKMTases also contain additional domains which enable them to respond to other PTMs or cellular signals. Here we show that the N-terminal WIYLD domain of the Arabidopsis SUVR4 HKMTase binds ubiquitin and that the SUVR4 product specificity shifts from di- to trimethylation in the presence of free ubiquitin, enabling conversion of H3K9me1 to H3K9me3 in vitro. Chromatin immunoprecipitation and immunocytological analysis showed that SUVR4 in vivo specifically converts H3K9me1 to H3K9me3 at transposons and pseudogenes and has a locus-specific repressive effect on the expression of such elements. Bisulfite sequencing indicates that this repression involves both DNA methylation-dependent and -independent mechanisms. Transcribed genes with high endogenous levels of H3K4me3, H3K9me3, and H2Bub1, but low H3K9me1, are generally unaffected by SUVR4 activity. Our results imply that SUVR4 is involved in the epigenetic defense mechanism by trimethylating H3K9 to suppress potentially harmful transposon activity.

  19. The Drosophila Su(var)3-7 gene is required for oogenesis and female fertility, genetically interacts with piwi and aubergine, but impacts only weakly transposon silencing.

    Science.gov (United States)

    Basquin, Denis; Spierer, Anne; Begeot, Flora; Koryakov, Dmitry E; Todeschini, Anne-Laure; Ronsseray, Stéphane; Vieira, Cristina; Spierer, Pierre; Delattre, Marion

    2014-01-01

    Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3-7 protein in Drosophila ovaries. We present evidences that Su(var)3-7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3-7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3-7 is required for genome integrity. Females homozygous for Su(var)3-7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3-7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3-7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3-7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3-7 and piwi or aubergine controls important developmental processes independently of transposon silencing.

  20. The Drosophila Su(var)3–7 Gene Is Required for Oogenesis and Female Fertility, Genetically Interacts with piwi and aubergine, but Impacts Only Weakly Transposon Silencing

    Science.gov (United States)

    Begeot, Flora; Koryakov, Dmitry E.; Todeschini, Anne-Laure; Ronsseray, Stéphane; Vieira, Cristina; Spierer, Pierre; Delattre, Marion

    2014-01-01

    Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3–7 protein in Drosophila ovaries. We present evidences that Su(var)3–7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3–7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3–7 is required for genome integrity. Females homozygous for Su(var)3–7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3–7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3–7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3–7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3–7 and piwi or aubergine controls important developmental processes independently of transposon silencing. PMID:24820312

  1. Genome-wide transposon mutagenesis of Proteus mirabilis: Essential genes, fitness factors for catheter-associated urinary tract infection, and the impact of polymicrobial infection on fitness requirements.

    Science.gov (United States)

    Armbruster, Chelsie E; Forsyth-DeOrnellas, Valerie; Johnson, Alexandra O; Smith, Sara N; Zhao, Lili; Wu, Weisheng; Mobley, Harry L T

    2017-06-01

    The Gram-negative bacterium Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs), which are often polymicrobial. Numerous prior studies have uncovered virulence factors for P. mirabilis pathogenicity in a murine model of ascending UTI, but little is known concerning pathogenesis during CAUTI or polymicrobial infection. In this study, we utilized five pools of 10,000 transposon mutants each and transposon insertion-site sequencing (Tn-Seq) to identify the full arsenal of P. mirabilis HI4320 fitness factors for single-species versus polymicrobial CAUTI with Providencia stuartii BE2467. 436 genes in the input pools lacked transposon insertions and were therefore concluded to be essential for P. mirabilis growth in rich medium. 629 genes were identified as P. mirabilis fitness factors during single-species CAUTI. Tn-Seq from coinfection with P. stuartii revealed 217/629 (35%) of the same genes as identified by single-species Tn-Seq, and 1353 additional factors that specifically contribute to colonization during coinfection. Mutants were constructed in eight genes of interest to validate the initial screen: 7/8 (88%) mutants exhibited the expected phenotypes for single-species CAUTI, and 3/3 (100%) validated the expected phenotypes for polymicrobial CAUTI. This approach provided validation of numerous previously described P. mirabilis fitness determinants from an ascending model of UTI, the discovery of novel fitness determinants specifically for CAUTI, and a stringent assessment of how polymicrobial infection influences fitness requirements. For instance, we describe a requirement for branched-chain amino acid biosynthesis by P. mirabilis during coinfection due to high-affinity import of leucine by P. stuartii. Further investigation of genes and pathways that provide a competitive advantage during both single-species and polymicrobial CAUTI will likely provide robust targets for therapeutic intervention to reduce P. mirabilis

  2. Stable, Nonviral Expression of Mutated Tumor Neoantigen-specific T-cell Receptors Using the Sleeping Beauty Transposon/Transposase System

    Science.gov (United States)

    Deniger, Drew C; Pasetto, Anna; Tran, Eric; Parkhurst, Maria R; Cohen, Cyrille J; Robbins, Paul F; Cooper, Laurence JN; Rosenberg, Steven A

    2016-01-01

    Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAKS2580F or ERBB2H473Y or the HLA-DQB*0601-restricted neoantigen ERBB2IPE805G were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRβ (mTCRβ) expression. Rapid expansion of mTCRβ+ T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27−CD45RA−) and less-differentiated (CD27+CD45RA+) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens. PMID:26945006

  3. Transposon fingerprinting using low coverage whole genome shotgun sequencing in cacao (Theobroma cacao L.) and related species.

    Science.gov (United States)

    Sveinsson, Saemundur; Gill, Navdeep; Kane, Nolan C; Cronk, Quentin

    2013-07-24

    Transposable elements (TEs) and other repetitive elements are a large and dynamically evolving part of eukaryotic genomes, especially in plants where they can account for a significant proportion of genome size. Their dynamic nature gives them the potential for use in identifying and characterizing crop germplasm. However, their repetitive nature makes them challenging to study using conventional methods of molecular biology. Next generation sequencing and new computational tools have greatly facilitated the investigation of TE variation within species and among closely related species. (i) We generated low-coverage Illumina whole genome shotgun sequencing reads for multiple individuals of cacao (Theobroma cacao) and related species. These reads were analysed using both an alignment/mapping approach and a de novo (graph based clustering) approach. (ii) A standard set of ultra-conserved orthologous sequences (UCOS) standardized TE data between samples and provided phylogenetic information on the relatedness of samples. (iii) The mapping approach proved highly effective within the reference species but underestimated TE abundance in interspecific comparisons relative to the de novo methods. (iv) Individual T. cacao accessions have unique patterns of TE abundance indicating that the TE composition of the genome is evolving actively within this species. (v) LTR/Gypsy elements are the most abundant, comprising c.10% of the genome. (vi) Within T. cacao the retroelement families show an order of magnitude greater sequence variability than the DNA transposon families. (vii) Theobroma grandiflorum has a similar TE composition to T. cacao, but the related genus Herrania is rather different, with LTRs making up a lower proportion of the genome, perhaps because of a massive presence (c. 20%) of distinctive low complexity satellite-like repeats in this genome. (i) Short read alignment/mapping to reference TE contigs provides a simple and effective method of investigating

  4. New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library.

    Science.gov (United States)

    Laia, Marcelo L; Moreira, Leandro M; Dezajacomo, Juliana; Brigati, Joice B; Ferreira, Cristiano B; Ferro, Maria I T; Silva, Ana C R; Ferro, Jesus A; Oliveira, Julio C F

    2009-01-16

    Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis. Through transposon insertion mutagenesis, 10,000 mutants of Xanthomonas citri subsp. citri strain 306 (Xcc) were obtained, and 3,300 were inoculated in Rangpur lime (Citrus limonia) leaves. Their ability to cause citrus canker was analyzed every 3 days until 21 days after inoculation; a set of 44 mutants showed altered virulence, with 8 presenting a complete loss of causing citrus canker symptoms. Sequencing of the insertion site in all 44 mutants revealed that 35 different ORFs were hit, since some ORFs were hit in more than one mutant, with mutants for the same ORF presenting the same phenotype. An analysis of these ORFs showed that some encoded genes were previously known as related to pathogenicity in phytobacteria and, more interestingly, revealed new genes never implicated with Xanthomonas pathogenicity before, including hypothetical ORFs. Among the 8 mutants with no canker symptoms are the hrpB4 and hrpX genes, two genes that belong to type III secretion system (TTSS), two hypothetical ORFS and, surprisingly, the htrA gene, a gene reported as involved with the virulence process in animal-pathogenic bacteria but not described as involved in phytobacteria virulence. Nucleic acid hybridization using labeled cDNA probes showed that some of the

  5. Generation of mariner-based transposon insertion mutant library of Bacillus sphaericus 2297 and investigation of genes involved in sporulation and mosquito-larvicidal crystal protein synthesis.

    Science.gov (United States)

    Wu, Yiming; Hu, Xiaomin; Ge, Yong; Zheng, Dasheng; Yuan, Zhiming

    2012-05-01

    Bacillus sphaericus has been used with great success in mosquito control programs worldwide. Under conditions of nutrient limitation, it undergoes sporulation via a series of well defined morphological stages. However, only a small number of genes involved in sporulation have been identified. To identify genes associated with sporulation, and to understand the relationship between sporulation and crystal protein synthesis, a random mariner-based transposon insertion mutant library of B. sphaericus strain 2297 was constructed and seven sporulation-defective mutants were selected. Sequencing of the DNA flanking of the transposon insertion identified several genes involved in sporulation. The morphologies of mutants were determined by electron microscopy and synthesis of crystal proteins was analyzed by SDS-PAGE and Western blot. Four mutants blocked at early stages of sporulation failed to produce crystal proteins and had lower larvicidal activity. However, the other three mutants were blocked at later stages and were able to form crystal proteins, and the larvicidal activity was similar to wild type. These results indicated that crystal protein synthesis in B. sphaericus is dependent on sporulation initiation. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  6. The Foldback-like element Galileo belongs to the P superfamily of DNA transposons and is widespread within the Drosophila genus.

    Science.gov (United States)

    Marzo, Mar; Puig, Marta; Ruiz, Alfredo

    2008-02-26

    Galileo is the only transposable element (TE) known to have generated natural chromosomal inversions in the genus Drosophila. It was discovered in Drosophila buzzatii and classified as a Foldback-like element because of its long, internally repetitive, terminal inverted repeats (TIRs) and lack of coding capacity. Here, we characterized a seemingly complete copy of Galileo from the D. buzzatii genome. It is 5,406 bp long, possesses 1,229-bp TIRs, and encodes a 912-aa transposase similar to those of the Drosophila melanogaster 1360 (Hoppel) and P elements. We also searched the recently available genome sequences of 12 Drosophila species for elements similar to Dbuz\\Galileo by using bioinformatic tools. Galileo was found in six species (ananassae, willistoni, peudoobscura, persimilis, virilis, and mojavensis) from the two main lineages within the Drosophila genus. Our observations place Galileo within the P superfamily of cut-and-paste transposons and extend considerably its phylogenetic distribution. The interspecific distribution of Galileo indicates an ancient presence in the genus, but the phylogenetic tree built with the transposase amino acid sequences contrasts significantly with that of the species, indicating lineage sorting and/or horizontal transfer events. Our results also suggest that Foldback-like elements such as Galileo may evolve from DNA-based transposon ancestors by loss of the transposase gene and disproportionate elongation of TIRs.

  7. Transposon mutagenesis of probiotic Lactobacillus casei identifies asnH, an asparagine synthetase gene involved in its immune-activating capacity.

    Science.gov (United States)

    Ito, Masahiro; Kim, Yun-Gi; Tsuji, Hirokazu; Takahashi, Takuya; Kiwaki, Mayumi; Nomoto, Koji; Danbara, Hirofumi; Okada, Nobuhiko

    2014-01-01

    Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.

  8. Transposon mutagenesis of probiotic Lactobacillus casei identifies asnH, an asparagine synthetase gene involved in its immune-activating capacity.

    Directory of Open Access Journals (Sweden)

    Masahiro Ito

    Full Text Available Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.

  9. Genetic structure of landraces in foxtail millet (Setaria italica (L.) P. Beauv.) revealed with transposon display and interpretation to crop evolution of foxtail millet.

    Science.gov (United States)

    Hirano, Ryoko; Naito, Ken; Fukunaga, Kenji; Watanabe, Kazuo N; Ohsawa, Ryo; Kawase, Makoto

    2011-06-01

    Although the origin and domestication process of foxtail millet (Setaria italica subsp. italica (L.) P. Beauv.) has been studied by several groups, the issue is still ambiguous. It is essential to resolve this issue by studying a large number of accessions with sufficient markers covering the entire genome. Genetic structures were analyzed by transposon display (TD) using 425 accessions of foxtail millet and 12 of the wild ancestor green foxtail (Setaria italica subsp. viridis (L.) P. Beauv.). We used three recently active transposons (TSI-1, TSI-7, and TSI-10) as genome-wide markers and succeeded in demonstrating geographical structures of the foxtail millet. A neighbor-joining dendrogram based on TD grouped the foxtail millet accessions into eight major clusters, each of which consisted of accessions collected from adjacent geographical areas. Eleven out of 12 green foxtail accessions were grouped separately from the clusters of foxtail millet. These results indicated strong regional differentiations and a long history of cultivation in each region. Furthermore, we discuss the relationship between foxtail millet and green foxtail and suggest a monophyletic origin of foxtail millet domestication.

  10. Diversity of the tetracycline resistance gene tet(M) and identification of Tn916- and Tn5801-like (Tn6014) transposons in Staphylococcus aureus from humans and animals

    DEFF Research Database (Denmark)

    de Vries, Lisbeth Elvira; Christensen, H.; Skov, R. L.

    2009-01-01

    To analyse the sequence diversity of the tetracycline resistance gene tet(M) in Staphylococcus aureus from humans and animals and to determine mobile elements associated with tet(M) in S. aureus. In total, 205 tetracycline-resistant isolates were screened for tet(M) by PCR. tet(M) genes were...... sequenced and compared with tet(M) deposited in GenBank. Based on phylogenetic analysis isolates were screened for Tn916- and Tn5801-like xis/int genes, and transposons were confirmed by linking PCR. spa typing was performed and selected isolates were used as donors in a filter mating experiment. Forty......-one isolates (21.3%, 60.7%, 2.6% and 4.4% of the human, pig, poultry and cattle isolates, respectively) were tet(M) positive. tet(M) was located on Tn5801-like and Tn916-like transposons in humans and on a specific Tn916-like element in animals. Human isolates were of different spa types (t034, t008, t037, t...

  11. A novel, simple, high-throughput method for isolation of genome-wide transposon insertion mutants of Escherichia coli K-12.

    Science.gov (United States)

    Miki, Takeyoshi; Yamamoto, Yoshihiro; Matsuda, Hideo

    2008-01-01

    We developed a novel, simple, high-throughput method for isolation of genome-wide transposon insertion mutants of Escherichia coli K-12. The basic idea of the method is to randomly disrupt the genes on the DNA fragments cloned on the Kohara library by inserting a mini-transposon first, and then transfer the disrupted genes from the lambda vector to the E. coli chromosome by homologous recombination. Using this method, we constructed a set of 8402 Km(r) cis-diploid mutants harboring a mini-Tn10 insertion mutation and the corresponding wild-type gene on a chromosome, as well as a set of 6954 haploid mutants derived from the cis-diploid mutants. The major advantage of the strategy used is that the indispensable genes or sites for growth can be identified. Preliminary results suggest that 415 open reading frames are indispensable for growth in E. coli cells. A total of 6404 haploid mutants were deposited to Genetic Strains Research Center, National Institute of Genetics, Japan (Chapter 26) and are available for public distribution upon request (http://shigen.lab.nig.ac.jp/ecoli/strain/nbrp/resource.jsp).

  12. Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites.

    Science.gov (United States)

    Jia, Xianbo; Lin, Xinjian; Chen, Jichen

    2017-11-02

    Current genome walking methods are very time consuming, and many produce non-specific amplification products. To amplify the flanking sequences that are adjacent to Tn5 transposon insertion sites in Serratia marcescens FZSF02, we developed a genome walking method based on TAIL-PCR. This PCR method added a 20-cycle linear amplification step before the exponential amplification step to increase the concentration of the target sequences. Products of the linear amplification and the exponential amplification were diluted 100-fold to decrease the concentration of the templates that cause non-specific amplification. Fast DNA polymerase with a high extension speed was used in this method, and an amplification program was used to rapidly amplify long specific sequences. With this linear and exponential TAIL-PCR (LETAIL-PCR), we successfully obtained products larger than 2 kb from Tn5 transposon insertion mutant strains within 3 h. This method can be widely used in genome walking studies to amplify unknown sequences that are adjacent to known sequences.

  13. Stable gene transfer of CCR5 and CXCR4 siRNAs by sleeping beauty transposon system to confer HIV-1 resistance

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    Akkina Ramesh

    2008-07-01

    Full Text Available Abstract Background Thus far gene therapy strategies for HIV/AIDS have used either conventional retroviral vectors or lentiviral vectors for gene transfer. Although highly efficient, their use poses a certain degree of risk in terms of viral mediated oncogenesis. Sleeping Beauty (SB transposon system offers a non-viral method of gene transfer to avoid this possible risk. With respect to conferring HIV resistance, stable knock down of HIV-1 coreceptors CCR5 and CXCR4 by the use of lentiviral vector delivered siRNAs has proved to be a promising strategy to protect cells from HIV-1 infection. In the current studies our aim is to evaluate the utility of SB system for stable gene transfer of CCR5 and CXCR4 siRNA genes to derive HIV resistant cells as a first step towards using this system for gene therapy. Results Two well characterized siRNAs against the HIV-1 coreceptors CCR5 and CXCR4 were chosen based on their previous efficacy for the SB transposon gene delivery. The siRNA transgenes were incorporated individually into a modified SB transfer plasmid containing a FACS sortable red fluorescence protein (RFP reporter and a drug selectable neomycin resistance gene. Gene transfer was achieved by co-delivery with a construct expressing a hyperactive transposase (HSB5 into the GHOST-R3/X4/R5 cell line, which expresses the major HIV receptor CD4 and and the co-receptors CCR5 and CXCR4. SB constructs expressing CCR5 or CXCR4 siRNAs were also transfected into MAGI-CCR5 or MAGI-CXCR4 cell lines, respectively. Near complete downregulation of CCR5 and CXCR4 surface expression was observed in transfected cells. During viral challenge with X4-tropic (NL4.3 or R5-tropic (BaL HIV-1 strains, the respective transposed cells showed marked viral resistance. Conclusion SB transposon system can be used to deliver siRNA genes for stable gene transfer. The siRNA genes against HIV-1 coreceptors CCR5 and CXCR4 are able to downregulate the respective cell surface proteins

  14. Expression of phage-transposons of Pseudomonas aeruginosa in cells of Pseudomonas putida PpGl. II. Zygotic induction, an essential condition for the emergence of defective lysogens

    Energy Technology Data Exchange (ETDEWEB)

    Gorbunova, S.A.; Akhverdyan, V.Z.; Krylov, V.N.

    1986-06-01

    The presence of a large number of clones, which have lost the ability to produce phase, is a feature of PpGl exconjugants, which have acquired markers of the hybrid plasmid containing the genome of the PAO1 phage transposon. Analysis of these clones indicates that they contain plasmids with different defects in the phage genome (point mutations and different deletion lengths which may have an effect on both the phage genome and also plasmid RP4). Mutations of a particular region are selected, the region of early prophage genes. The process of zygotic induction is an essential condition for the arisal of mutants (including deletion mutants). The results of the experiment, which was based on the Luria-Delbruik test, showed that a significant proportion of the mutations, including deletion mutations, arise in the recipient cells PpGl.

  15. Characterization of Tn3000, a Transposon Responsible for blaNDM-1 Dissemination among Enterobacteriaceae in Brazil, Nepal, Morocco, and India.

    Science.gov (United States)

    Campos, Juliana Coutinho; da Silva, Maria José Félix; dos Santos, Paulo Roberto Nascimento; Barros, Elaine Menezes; Pereira, Mayne de Oliveira; Seco, Bruna Mara Silva; Magagnin, Cibele Massotti; Leiroz, Leonardo Kalab; de Oliveira, Théo Gremen Mimary; de Faria-Júnior, Célio; Cerdeira, Louise Teixeira; Barth, Afonso Luís; Sampaio, Suely Carlos Ferreira; Zavascki, Alexandre Prehn; Poirel, Laurent; Sampaio, Jorge Luiz Mello

    2015-12-01

    In Enterobacteriaceae, the blaNDM genes have been found in many different genetic contexts, and a wide diversity of plasmid scaffolds bearing those genes has been found. In August 2013, we identified NDM-1-producing Escherichia coli and Enterobacter hormaechei strains from a single rectal swab sample from a patient hospitalized in Rio de Janeiro, Brazil, who had no history of travel abroad. Complete DNA sequencing using the Illumina platform and annotation of the two plasmids harboring the blaNDM-1 gene, one from each strain, showed that they belonged to incompatibility groups IncFIIK and IncX3 and harbored a novel transposon named Tn3000. Similar genetic structures have been identified among other isolates in Brazil but also on plasmids from other continents. Our findings suggest that the blaNDM-1 gene may be transmitted by Tn3000 in different parts of the world. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. High copy number of highly similar mariner-like transposons in planarian (Platyhelminthe): evidence for a trans-phyla horizontal transfer.

    Science.gov (United States)

    Garcia-Fernàndez, J; Bayascas-Ramírez, J R; Marfany, G; Muñoz-Mármol, A M; Casali, A; Baguñà, J; Saló, E

    1995-05-01

    Several DNA sequences similar to the mariner element were isolated and characterized in the platyhelminthe Dugesia (Girardia) tigrina. They were 1,288 bp long, flanked by two 32 bp-inverted repeats, and contained a single 339 amino acid open-reading frame (ORF) encoding the transposase. The number of copies of this element is approximately 8,000 per haploid genome, constituting a member of the middle-repetitive DNA of Dugesia tigrina. Sequence analysis of several elements showed a high percentage of conservation between the different copies. Most of them presented an intact ORF and the standard signals of actively expressed genes, which suggests that some of them are or have recently been functional transposons. The high degree of similarity shared with other mariner elements from some arthropods, together with the fact that this element is undetectable in other planarian species, strongly suggests a case of horizontal transfer between these two distant phyla.

  17. A Traceless Selection: Counter-selection System That Allows Efficient Generation of Transposon and CRISPR-modified T-cell Products

    Directory of Open Access Journals (Sweden)

    Riccardo Mezzadra

    2016-01-01

    Full Text Available Recent years have seen major breakthroughs in genome-engineering systems, such as transposon-mediated gene delivery systems and CRISPR-Cas9-mediated genome-editing tools. In these systems, transient expression of auxiliary genes is responsible for permanent genomic modification. For both systems, it would be valuable to select for cells that are likely to undergo stable genome modification. Importantly, in particular for clinical applications of genome-engineered cell products, it will also be of importance to remove those cells that, due to random vector integration, display an unwanted stable expression of the auxiliary gene. Here, we develop a traceless selection system that on the one hand allows efficient enrichment of modified cells, and on the other hand can be used to select against cells that retain expression of the auxiliary gene. The value of this system to produce highly enriched-auxiliary gene-free cell products is demonstrated.

  18. A transposon mutant library of Bacillus cereus ATCC 10987 reveals novel genes required for biofilm formation and implicates motility as an important factor for pellicle-biofilm formation.

    Science.gov (United States)

    Okshevsky, Mira; Louw, Matilde Greve; Lamela, Elena Otero; Nilsson, Martin; Tolker-Nielsen, Tim; Meyer, Rikke Louise

    2018-04-01

    Bacillus cereus is one of the most common opportunistic pathogens causing foodborne illness, as well as a common source of contamination in the dairy industry. B. cereus can form robust biofilms on food processing surfaces, resulting in food contamination due to shedding of cells and spores. Despite the medical and industrial relevance of this species, the genetic basis of biofilm formation in B. cereus is not well studied. In order to identify genes required for biofilm formation in this bacterium, we created a library of 5000 +  transposon mutants of the biofilm-forming strain B. cereusATCC 10987, using an unbiased mariner transposon approach. The mutant library was screened for the ability to form a pellicle biofilm at the air-media interface, as well as a submerged biofilm at the solid-media interface. A total of 91 genes were identified as essential for biofilm formation. These genes encode functions such as chemotaxis, amino acid metabolism and cellular repair mechanisms, and include numerous genes not previously known to be required for biofilm formation. Although the majority of disrupted genes are not directly responsible for motility, further investigations revealed that the vast majority of the biofilm-deficient mutants were also motility impaired. This observation implicates motility as a pivotal factor in the formation of a biofilm by B. cereus. These results expand our knowledge of the fundamental molecular mechanisms of biofilm formation by B. cereus. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  19. Complete nucleotide sequence of CTX-M-15-plasmids from clinical Escherichia coli isolates: insertional events of transposons and insertion sequences.

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    Annemieke Smet

    Full Text Available BACKGROUND: CTX-M-producing Escherichia coli strains are regarded as major global pathogens. METHODOLOGY/PRINCIPAL FINDINGS: The nucleotide sequence of three plasmids (pEC_B24: 73801-bp; pEC_L8: 118525-bp and pEC_L46: 144871-bp from Escherichia coli isolates obtained from patients with urinary tract infections and one plasmid (pEC_Bactec: 92970-bp from an Escherichia coli strain isolated from the joint of a horse with arthritis were determined. Plasmid pEC_Bactec belongs to the IncI1 group and carries two resistance genes: bla(TEM-1 and bla(CTX-M-15. It shares more than 90% homology with a previously published bla(CTX-M-plasmid from E. coli of human origin. Plasmid pEC_B24 belongs to the IncFII group whereas plasmids pEC_L8 and pEC_L46 represent a fusion of two replicons of type FII and FIA. On the pEC_B24 backbone, two resistance genes, bla(TEM-1 and bla(CTX-M-15, were found. Six resistance genes, bla(TEM-1, bla(CTX-M-15, bla(OXA-1, aac6'-lb-cr, tetA and catB4, were detected on the pEC_L8 backbone. The same antimicrobial drug resistance genes, with the exception of tetA, were also identified on the pEC_L46 backbone. Genome analysis of all 4 plasmids studied provides evidence of a seemingly frequent transposition event of the bla(CTX-M-15-ISEcp1 element. This element seems to have a preferred insertion site at the tnpA gene of a bla(TEM-carrying Tn3-like transposon, the latter itself being inserted by a transposition event. The IS26-composite transposon, which contains the bla(OXA-1, aac6'-lb-cr and catB4 genes, was inserted into plasmids pEC_L8 and pEC_L46 by homologous recombination rather than a transposition event. Results obtained for pEC_L46 indicated that IS26 also plays an important role in structural rearrangements of the plasmid backbone and seems to facilitate the mobilisation of fragments from other plasmids. CONCLUSIONS: Collectively, these data suggests that IS26 together with ISEcp1 could play a critical role in the evolution of

  20. Wide Awake and Ready to Move: 20 Years of Non-Viral Therapeutic Genome Engineering with the Sleeping Beauty Transposon System.

    Science.gov (United States)

    Hodge, Russ; Narayanavari, Suneel A; Izsvák, Zsuzsanna; Ivics, Zoltán

    2017-10-01

    Gene therapies will only become a widespread tool in the clinical treatment of human diseases with the advent of gene transfer vectors that integrate genetic information stably, safely, effectively, and economically. Two decades after the discovery of the Sleeping Beauty (SB) transposon, it has been transformed into a vector system that is fulfilling these requirements. SB may well overcome some of the limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are being used in the majority of ongoing clinical trials. The SB system has achieved a high level of stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, representing crucial steps that may permit its clinical use in the near future. This article reviews the most important aspects of SB as a tool for gene therapy, including aspects of its vectorization and genomic integration. As an illustration, the clinical development of the SB system toward gene therapy of age-related macular degeneration and cancer immunotherapy is highlighted.

  1. Combination of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 technique with the piggybac transposon system for mouse in utero electroporation to study cortical development.

    Science.gov (United States)

    Cheng, Man; Jin, Xubin; Mu, Lili; Wang, Fangyu; Li, Wei; Zhong, Xiaoling; Liu, Xuan; Shen, Wenchen; Liu, Ying; Zhou, Yan

    2016-09-01

    In utero electroporation (IUE) is commonly used to study cortical development of cerebrum by downregulating or overexpressing genes of interest in neural progenitor cells (NPCs) of small mammals. However, exogenous plasmids are lost or diluted over time. Furthermore, gene knockdown based on short-hairpin RNAs may exert nonspecific effects that lead to aberrant neuronal migration. Genomic engineering by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has great research and therapeutic potentials. Here we integrate the CRISPR/Cas9 components into the piggyBac (PB) transposon system (the CRISPR/Cas9-PB toolkit) for cortical IUEs. The mouse Sry-related HMG box-2 (Sox2) gene was selected as the target for its application. Most transduced cortical NPCs were depleted of SOX2 protein as early as 3 days post-IUE, whereas expressions of SOX1 and PAX6 remained intact. Furthermore, both the WT Cas9 and the D10A nickase mutant Cas9n showed comparable knockout efficiency. Transduced cortical cells were purified with fluorescence-activated cell sorting, and effective gene editing at the Sox2 loci was confirmed. Thus, application of the CRISPR/Cas9-PB toolkit in IUE is a promising strategy to study gene functions in cortical NPCs and their progeny. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. Genetic analysis of attTn7, the transposon Tn7 attachment site in Escherichia coli, using a novel M13-based transduction assay.

    Science.gov (United States)

    Qadri, M I; Flores, C C; Davis, A J; Lichtenstein, C P

    1989-05-05

    The large (14 kb; kb = 10(3) bases) bacterial transposon, Tn7 (encoding resistance to trimethoprim and streptomycin/spectinomycin), has unusual properties. Like other elements, Tn7 transposes with low efficiency and low target-site specificity, but Tn7 also transposes, with high frequency in a unique orientation, to a preferred "attachment" site, called attTn7, in the Escherichia coli chromosome and similarly into plasmids containing attTn7. We developed a novel bacteriophage M13-based assay system to measure the transposition frequency of Tn7 to M13mp phage vectors containing attTn7 on a cloned 1 kb fragment of chromosomal DNA. Phage harvested from a Tn7 donor strain were used to infect recipient bacteria with selection for trimethoprim resistance. Transposition frequency, expressed as the number of trimethoprim-resistant colonies per plaque-forming unit, was found to be approximately 10(-4) to M13mp::attTn7, in contrast to 10(-10) to M13mp recombinants with approximately 1 kb insertions of other, "generic brand", DNA. By deletion analysis of M13mp::attTn7, we show that attTn7 is contained within a 64 base-pair region; sequences adjacent to the actual insertion site and encoding the carboxy terminus of the glmS gene are required. This assay also provided evidence for transposition immunity conferred by the right end of Tn7.

  3. A new biotype of Fusarium oxysporum f. sp. lycopersici race 2 emerged by a transposon-driven mutation of avirulence gene AVR1.

    Science.gov (United States)

    Kashiwa, Takeshi; Suzuki, Tatsuya; Sato, Akira; Akai, Kotaro; Teraoka, Tohru; Komatsu, Ken; Arie, Tsutomu

    2016-07-01

    Emergence of races in Fusarium oxysporum f. sp. lycopersici (Fol) is caused by loss or mutation of at least one avirulence (AVR) gene. The product of AVR1 is a small protein (Avr1) secreted by Fol in tomato xylem sap during infection. This protein triggers Fol race 1 specific resistance (I) in tomato, indicating that AVR1 is an AVR gene. Deletion of AVR1 in race 1 resulted in the emergence of race 2, and an additional mutation in AVR2 generated race 3. Previously, we reported a new biotype of race 3, KoChi-1, in which AVR1 was truncated by a transposon Hormin, which suggested a new route to evolution of races in Fol However, to date no race 2 isolate carrying Hormin-truncated AVR1 has been reported. In this report, we describe such isolates, represented by Chiba-5, in which Hormin insertion occurred in AVR1 at a position different from that in KoChi-1. AVR1 truncation in both isolates resulted in production of defective Avr1 proteins. Chiba-5 and KoChi-1 belong to different phylogenetic clades, A1 and A2, respectively, suggesting that insertion of Hormin in AVR1 in Chiba-5 and KoChi-1 occurred as independent evolutionary events. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications.

    Science.gov (United States)

    Kolacsek, Orsolya; Pergel, Enikő; Varga, Nóra; Apáti, Ágota; Orbán, Tamás I

    2017-01-20

    There are numerous applications of quantitative PCR for both diagnostic and basic research. As in many other techniques the basis of quantification is that comparisons are made between different (unknown and known or reference) specimens of the same entity. When the aim is to compare real quantities of different species in samples, one cannot escape their separate precise absolute quantification. We have established a simple and reliable method for this purpose (Ct shift method) which combines the absolute and the relative approach. It requires a plasmid standard containing both sequences of amplicons to be compared (e.g. the target of interest and the endogenous control). It can serve as a reference sample with equal copies of templates for both targets. Using the ΔΔCt formula we can quantify the exact ratio of the two templates in each unknown sample. The Ct shift method has been successfully applied for transposon gene copy measurements, as well as for comparison of different mRNAs in cDNA samples. This study provides the proof of concept and introduces some potential applications of the method; the absolute nature of results even without the need for real reference samples can contribute to the universality of the method and comparability of different studies. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Copy number of the transposon, Pokey, in rDNA is positively correlated with rDNA copy number in Daphnia obtuse [corrected].

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    Kaitlynn LeRiche

    Full Text Available Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL initiated from a single D. obtusa female. The change in direction and magnitude of Pokey and rRNA gene number did not show a consistent pattern across ∼ 87 generations in the MAL; however, Pokey and rRNA gene number changed in concert. PokeyA and 28S gene number were positively correlated in the isolates from both natural populations and the MAL. PokeyB number was much lower than PokeyA in both MAL and natural population isolates, and showed no correlation with 28S gene number. Preliminary analysis did not detect PokeyB outside rDNA in any isolates and detected only 0 to 4 copies of PokeyA outside rDNA indicating that Pokey may be primarily an rDNA element in D. obtusa. The recombination rate in this species is high and the average size of the rDNA locus is about twice as large as that in other Daphnia species such as D. pulicaria and D. pulex, which may have facilitated expansion of PokeyA to much higher numbers in D. obtusa rDNA than these other species.

  6. In silico polymorphism analysis for the development of simple sequence repeat and transposon markers and construction of linkage map in cultivated peanut

    Directory of Open Access Journals (Sweden)

    Shirasawa Kenta

    2012-06-01

    Full Text Available Abstract Background Peanut (Arachis hypogaea is an autogamous allotetraploid legume (2n = 4x = 40 that is widely cultivated as a food and oil crop. More than 6,000 DNA markers have been developed in Arachis spp., but high-density linkage maps useful for genetics, genomics, and breeding have not been constructed due to extremely low genetic diversity. Polymorphic marker loci are useful for the construction of such high-density linkage maps. The present study used in silico analysis to develop simple sequence repeat-based and transposon-based markers. Results The use of in silico analysis increased the efficiency of polymorphic marker development by more than 3-fold. In total, 926 (34.2% of 2,702 markers showed polymorphisms between parental lines of the mapping population. Linkage analysis of the 926 markers along with 253 polymorphic markers selected from 4,449 published markers generated 21 linkage groups covering 2,166.4 cM with 1,114 loci. Based on the map thus produced, 23 quantitative trait loci (QTLs for 15 agronomical traits were detected. Another linkage map with 326 loci was also constructed and revealed a relationship between the genotypes of the FAD2 genes and the ratio of oleic/linoleic acid in peanut seed. Conclusions In silico analysis of polymorphisms increased the efficiency of polymorphic marker development, and contributed to the construction of high-density linkage maps in cultivated peanut. The resultant maps were applicable to QTL analysis. Marker subsets and linkage maps developed in this study should be useful for genetics, genomics, and breeding in Arachis. The data are available at the Kazusa DNA Marker Database (http://marker.kazusa.or.jp.

  7. A comprehensive analysis of in vitro and in vivo genetic fitness of Pseudomonas aeruginosa using high-throughput sequencing of transposon libraries.

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    David Skurnik

    Full Text Available High-throughput sequencing of transposon (Tn libraries created within entire genomes identifies and quantifies the contribution of individual genes and operons to the fitness of organisms in different environments. We used insertion-sequencing (INSeq to analyze the contribution to fitness of all non-essential genes in the chromosome of Pseudomonas aeruginosa strain PA14 based on a library of ∼300,000 individual Tn insertions. In vitro growth in LB provided a baseline for comparison with the survival of the Tn insertion strains following 6 days of colonization of the murine gastrointestinal tract as well as a comparison with Tn-inserts subsequently able to systemically disseminate to the spleen following induction of neutropenia. Sequencing was performed following DNA extraction from the recovered bacteria, digestion with the MmeI restriction enzyme that hydrolyzes DNA 16 bp away from the end of the Tn insert, and fractionation into oligonucleotides of 1,200-1,500 bp that were prepared for high-throughput sequencing. Changes in frequency of Tn inserts into the P. aeruginosa genome were used to quantify in vivo fitness resulting from loss of a gene. 636 genes had <10 sequencing reads in LB, thus defined as unable to grow in this medium. During in vivo infection there were major losses of strains with Tn inserts in almost all known virulence factors, as well as respiration, energy utilization, ion pumps, nutritional genes and prophages. Many new candidates for virulence factors were also identified. There were consistent changes in the recovery of Tn inserts in genes within most operons and Tn insertions into some genes enhanced in vivo fitness. Strikingly, 90% of the non-essential genes were required for in vivo survival following systemic dissemination during neutropenia. These experiments resulted in the identification of the P. aeruginosa strain PA14 genes necessary for optimal survival in the mucosal and systemic environments of a mammalian

  8. Development of Transgenic Cloned Pig Models of Skin Inflammation by DNA Transposon-Directed Ectopic Expression of Human β1 and α2 Integrin

    Science.gov (United States)

    Staunstrup, Nicklas Heine; Madsen, Johannes; Primo, Maria Nascimento; Li, Juan; Liu, Ying; Kragh, Peter M.; Li, Rong; Schmidt, Mette; Purup, Stig; Dagnæs-Hansen, Frederik; Svensson, Lars; Petersen, Thomas K.; Callesen, Henrik; Bolund, Lars; Mikkelsen, Jacob Giehm

    2012-01-01

    Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to severe skin damage

  9. Tol2 transposon-mediated transgenesis in the Midas cichlid (Amphilophus citrinellus) - towards understanding gene function and regulatory evolution in an ecological model system for rapid phenotypic diversification.

    Science.gov (United States)

    Kratochwil, Claudius F; Sefton, Maggie M; Liang, Yipeng; Meyer, Axel

    2017-11-23

    The Midas cichlid species complex (Amphilophus spp.) is widely known among evolutionary biologists as a model system for sympatric speciation and adaptive phenotypic divergence within extremely short periods of time (a few hundred generations). The repeated parallel evolution of adaptive phenotypes in this radiation, combined with their near genetic identity, makes them an excellent model for studying phenotypic diversification. While many ecological and evolutionary studies have been performed on Midas cichlids, the molecular basis of specific phenotypes, particularly adaptations, and their underlying coding and cis-regulatory changes have not yet been studied thoroughly. For the first time in any New World cichlid, we use Tol2 transposon-mediated transgenesis in the Midas cichlid (Amphilophus citrinellus). By adapting existing microinjection protocols, we established an effective protocol for transgenesis in Midas cichlids. Embryos were injected with a Tol2 plasmid construct that drives enhanced green fluorescent protein (eGFP) expression under the control of the ubiquitin promoter. The transgene was successfully integrated into the germline, driving strong ubiquitous expression of eGFP in the first transgenic Midas cichlid line. Additionally, we show transient expression of two further transgenic constructs, ubiquitin::tdTomato and mitfa::eGFP. Transgenesis in Midas cichlids will facilitate further investigation of the genetic basis of species-specific traits, many of which are adaptations. Transgenesis is a versatile tool not only for studying regulatory elements such as promoters and enhancers, but also for testing gene function through overexpression of allelic gene variants. As such, it is an important first step in establishing the Midas cichlid as a powerful model for studying adaptive coding and non-coding changes in an ecological and evolutionary context.

  10. Essential Genes for In Vitro Growth of the Endophyte Herbaspirillum seropedicae SmR1 as Revealed by Transposon Insertion Site Sequencing.

    Science.gov (United States)

    Rosconi, Federico; de Vries, Stefan P W; Baig, Abiyad; Fabiano, Elena; Grant, Andrew J

    2016-11-15

    The interior of plants contains microorganisms (referred to as endophytes) that are distinct from those present at the root surface or in the surrounding soil. Herbaspirillum seropedicae strain SmR1, belonging to the betaproteobacteria, is an endophyte that colonizes crops, including rice, maize, sugarcane, and sorghum. Different approaches have revealed genes and pathways regulated during the interactions of H. seropedicae with its plant hosts. However, functional genomic analysis of transposon (Tn) mutants has been hampered by the lack of genetic tools. Here we successfully employed a combination of in vivo high-density mariner Tn mutagenesis and targeted Tn insertion site sequencing (Tn-seq) in H. seropedicae SmR1. The analysis of multiple gene-saturating Tn libraries revealed that 395 genes are essential for the growth of H. seropedicae SmR1 in tryptone-yeast extract medium. A comparative analysis with the Database of Essential Genes (DEG) showed that 25 genes are uniquely essential in H. seropedicae SmR1. The Tn mutagenesis protocol developed and the gene-saturating Tn libraries generated will facilitate elucidation of the genetic mechanisms of the H. seropedicae endophytic lifestyle. A focal point in the study of endophytes is the development of effective biofertilizers that could help to reduce the input of agrochemicals in croplands. Besides the ability to promote plant growth, a good biofertilizer should be successful in colonizing its host and competing against the native microbiota. By using a systematic Tn-based gene-inactivation strategy and massively parallel sequencing of Tn insertion sites (Tn-seq), it is possible to study the fitness of thousands of Tn mutants in a single experiment. We have applied the combination of these techniques to the plant-growth-promoting endophyte Herbaspirillum seropedicae SmR1. The Tn mutant libraries generated will enable studies into the genetic mechanisms of H. seropedicae-plant interactions. The approach that we

  11. The complete genome sequencing of Prevotella intermedia strain OMA14 and a subsequent fine-scale, intra-species genomic comparison reveal an unusual amplification of conjugative and mobile transposons and identify a novel Prevotella-lineage-specific repeat.

    Science.gov (United States)

    Naito, Mariko; Ogura, Yoshitoshi; Itoh, Takehiko; Shoji, Mikio; Okamoto, Masaaki; Hayashi, Tetsuya; Nakayama, Koji

    2016-02-01

    Prevotella intermedia is a pathogenic bacterium involved in periodontal diseases. Here, we present the complete genome sequence of a clinical strain, OMA14, of this bacterium along with the results of comparative genome analysis with strain 17 of the same species whose genome has also been sequenced, but not fully analysed yet. The genomes of both strains consist of two circular chromosomes: the larger chromosomes are similar in size and exhibit a high overall linearity of gene organizations, whereas the smaller chromosomes show a significant size variation and have undergone remarkable genome rearrangements. Unique features of the Pre. intermedia genomes are the presence of a remarkable number of essential genes on the second chromosomes and the abundance of conjugative and mobilizable transposons (CTns and MTns). The CTns/MTns are particularly abundant in the second chromosomes, involved in its extensive genome rearrangement, and have introduced a number of strain-specific genes into each strain. We also found a novel 188-bp repeat sequence that has been highly amplified in Pre. intermedia and are specifically distributed among the Pre. intermedia-related species. These findings expand our understanding of the genetic features of Pre. intermedia and the roles of CTns and MTns in the evolution of bacteria. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  12. A Constitutively Mannose-Sensitive Agglutinating Salmonella enterica subsp. enterica Serovar Typhimurium Strain, Carrying a Transposon in the Fimbrial Usher Gene stbC, Exhibits Multidrug Resistance and Flagellated Phenotypes

    Directory of Open Access Journals (Sweden)

    Kuan-Hsun Wu

    2012-01-01

    Full Text Available Static broth culture favors Salmonella enterica subsp. enterica serovar Typhimurium to produce type 1 fimbriae, while solid agar inhibits its expression. A transposon inserted in stbC, which would encode an usher for Stb fimbriae of a non-flagellar Salmonella enterica subsp. enterica serovar Typhimurium LB5010 strain, conferred it to agglutinate yeast cells on both cultures. RT-PCR revealed that the expression of the fimbrial subunit gene fimA, and fimZ, a regulatory gene of fimA, were both increased in the stbC mutant when grown on LB agar; fimW, a repressor gene of fimA, exhibited lower expression. Flagella were observed in the stbC mutant and this phenotype was correlated with the motile phenotype. Microarray data and RT-PCR indicated that the expression of three genes, motA, motB, and cheM, was enhanced in the stbC mutant. The stbC mutant was resistant to several antibiotics, consistent with the finding that expression of yhcQ and ramA was enhanced. A complementation test revealed that transforming a recombinant plasmid possessing the stbC restored the mannose-sensitive agglutination phenotype to the stbC mutant much as that in the parental Salmonella enterica subsp. enterica serovar Typhimurium LB5010 strain, indicating the possibility of an interplay of different fimbrial systems in coordinating their expression.

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

    Science.gov (United States)

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

    2017-02-01

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

  14. Functional genomics strategies with transposons in rice

    NARCIS (Netherlands)

    Greco, R.

    2003-01-01

    Rice is a major staple food crop and a recognizedmonocotylenedousmodel plant from which gene function discovery is projected to contribute to improvements in a variety of cereals like wheat and maize. The recent release of rough drafts of the rice genome sequence for public

  15. UV-B damage amplified by transposons in maize

    International Nuclear Information System (INIS)

    Walbot, V.

    1999-01-01

    While absorbing visible light energy for photosynthesis, plants are unavoidably exposed to ultraviolet radiation, which is particularly harmful at shorter wavelengths (UV-B radiation). Ozone depletion in the atmosphere means that plants receive episodic or steadily increasing doses of UV-B, which damages their photosynthetic reaction centres, crosslinks cellular proteins, and induces mutagenic DNA lesions. Plant adaptive mechanisms of shielding and repair are therefore critical to survival — for example, somatic tissues of maize and Arabidopsis defective in phenolic sunscreen pigments incur increased DNA damage, and mutants defective in DNA repair are killed by UV-B

  16. Horizontal transfer of transposons between and within crustaceans and insects.

    Science.gov (United States)

    Dupeyron, Mathilde; Leclercq, Sébastien; Cerveau, Nicolas; Bouchon, Didier; Gilbert, Clément

    2014-01-29

    Horizontal transfer of transposable elements (HTT) is increasingly appreciated as an important source of genome and species evolution in eukaryotes. However, our understanding of HTT dynamics is still poor in eukaryotes because the diversity of species for which whole genome sequences are available is biased and does not reflect the global eukaryote diversity. In this study we characterized two Mariner transposable elements (TEs) in the genome of several terrestrial crustacean isopods, a group of animals particularly underrepresented in genome databases. The two elements have a patchy distribution in the arthropod tree and they are highly similar (>93% over the entire length of the element) to insect TEs (Diptera and Hymenoptera), some of which were previously described in Ceratitis rosa (Crmar2) and Drosophila biarmipes (Mariner-5_Dbi). In addition, phylogenetic analyses and comparisons of TE versus orthologous gene distances at various phylogenetic levels revealed that the taxonomic distribution of the two elements is incompatible with vertical inheritance. We conclude that the two Mariner TEs each underwent at least three HTT events. Both elements were transferred once between isopod crustaceans and insects and at least once between isopod crustacean species. Crmar2 was also transferred between tephritid and drosophilid flies and Mariner-5 underwent HT between hymenopterans and dipterans. We demonstrate that these various HTTs took place recently (most likely within the last 3 million years), and propose iridoviruses and/or Wolbachia endosymbionts as potential vectors of these transfers.

  17. The concerted impact of domestication and transposon insertions on methylation patterns between dogs and gray wolves

    Science.gov (United States)

    Koch, Ilana Janowitz; Clark, Michelle M.; Thompson, Michael J.; Deere-Machemer, Kerry A.; Wang, Jun; Duarte, Lionel; Gnanadesikan, Gitanjali E.; McCoy, Eskender L.; Rubbi, Liudmilla; Stahler, Daniel R.; Pellegrini, Matteo; Ostrander, Elaine A.; Wayne, Robert K.; Sinsheimer, Janet S.; vonHoldt, Bridgett M.

    2015-01-01

    The process of domestication can exert intense trait-targeted selection on genes and regulatory regions. Specifically, rapid shifts in the structure and sequence of genomic regulatory elements could provide an explanation for the extensive, and sometimes extreme, variation in phenotypic traits observed in domesticated species. Here, we explored methylation differences from >24,000 cytosines distributed across the genomes of the domesticated dog (Canis familiaris) and the gray wolf (C. lupus). PCA and model-based cluster analyses identified two primary groups, domestic versus wild canids. A scan for significantly differentially methylated sites (DMSs) revealed species-specific patterns at 68 sites after correcting for cell heterogeneity, with weak yet significant hyper-methylation typical of purebred dogs when compared to wolves (59% and 58%, p66%) of differentially methylated regions contained or were associated with repetitive elements, indicative of a genotype-mediated trend. However, DMSs were also often linked to functionally relevant genes (e.g. neurotransmitters). Finally, we utilized known genealogical relationships among Yellowstone wolves to survey transmission stability of methylation marks, from which we found a substantial fraction that demonstrated high heritability (both H2 and h2>0.99). These analyses provide a unique epigenetic insight into the molecular consequences of recent selection and radiation of our most ancient domesticated companion, the dog. These findings suggest selection has acted on methylation patterns, providing a new genomic perspective on phenotypic diversification in domesticated species. PMID:27112634

  18. The concerted impact of domestication and transposon insertions on methylation patterns between dogs and grey wolves.

    Science.gov (United States)

    Janowitz Koch, Ilana; Clark, Michelle M; Thompson, Michael J; Deere-Machemer, Kerry A; Wang, Jun; Duarte, Lionel; Gnanadesikan, Gitanjali E; McCoy, Eskender L; Rubbi, Liudmilla; Stahler, Daniel R; Pellegrini, Matteo; Ostrander, Elaine A; Wayne, Robert K; Sinsheimer, Janet S; vonHoldt, Bridgett M

    2016-04-01

    The process of domestication can exert intense trait-targeted selection on genes and regulatory regions. Specifically, rapid shifts in the structure and sequence of genomic regulatory elements could provide an explanation for the extensive, and sometimes extreme, variation in phenotypic traits observed in domesticated species. Here, we explored methylation differences from >24 000 cytosines distributed across the genomes of the domesticated dog (Canis familiaris) and the grey wolf (Canis lupus). PCA and model-based cluster analyses identified two primary groups, domestic vs. wild canids. A scan for significantly differentially methylated sites (DMSs) revealed species-specific patterns at 68 sites after correcting for cell heterogeneity, with weak yet significant hypermethylation typical of purebred dogs when compared to wolves (59% and 58%, P 66%) of differentially methylated regions contained or were associated with repetitive elements, indicative of a genotype-mediated trend. However, DMSs were also often linked to functionally relevant genes (e.g. neurotransmitters). Finally, we utilized known genealogical relationships among Yellowstone wolves to survey transmission stability of methylation marks, from which we found a substantial fraction that demonstrated high heritability (both H(2) and h(2 ) > 0.99). These analyses provide a unique epigenetic insight into the molecular consequences of recent selection and radiation of our most ancient domesticated companion, the dog. These findings suggest selection has acted on methylation patterns, providing a new genomic perspective on phenotypic diversification in domesticated species. © 2015 John Wiley & Sons Ltd.

  19. A four-element based transposon system for allele specific tagging ...

    Indian Academy of Sciences (India)

    The four-element based construct would include both Ds and dSpm along with relevant marker genes and .... subsequent generations after transposition has taken place so as to ..... Cardon G H, Frey M, Seadler H and Gierl A 1993 Definition.

  20. Quetzal: a transposon of the Tc1 family in the mosquito Anopheles albimanus.

    Science.gov (United States)

    Ke, Z; Grossman, G L; Cornel, A J; Collins, F H

    1996-10-01

    A member of the Tc1 family of transposable elements has been identified in the Central and South American mosquito Anopheles albimanus. The full-length Quetzal element is 1680 base pairs (bp) in length, possesses 236 bp inverted terminal repeats (ITRs), and has a single open reading frame (ORF) with the potential of encoding a 341-amino-acid (aa) protein that is similar to the transposases of other members of the Tc1 family, particularly elements described from three different Drosophila species. The approximately 10-12 copies per genome of Quetzal are found in the euchromatin of all three chromosomes of A. albimanus. One full-length clone, Que27, appears capable of encoding a complete transposase and may represent a functional copy of this element.

  1. New transposon tools tailored for metabolic engineering of Gram-negative microbial cell factories

    Directory of Open Access Journals (Sweden)

    Esteban eMartínez-García

    2014-10-01

    Full Text Available Re-programming microorganisms to modify their existing functions and/or to bestow bacteria with entirely new-to-Nature tasks have largely relied so far on specialized molecular biology tools. Such endeavors are not only relevant in the burgeoning metabolic engineering arena, but also instrumental to explore the functioning of complex regulatory networks from a fundamental point of view. À la carte modification of bacterial genomes thus calls for novel tools to make genetic manipulations easier. We propose the use of a series of new broad-host-range mini-Tn5 vectors, termed pBAMDs, for the delivery of gene(s into the chromosome of Gram-negative bacteria and for generating saturated mutagenesis libraries in gene function studies. These delivery vectors endow the user with the possibility of easy cloning and subsequent insertion of functional cargoes with three different antibiotic resistance markers (kanamycin, streptomycin, and gentamicin. After validating the pBAMD vectors in the environmental bacterium Pseudomonas putida KT2440, their use was also illustrated by inserting the entire poly(3-hydroxybutyrate (PHB synthesis pathway from Cupriavidus necator in the chromosome of a phosphotransacetylase mutant of Escherichia coli. PHB is a completely biodegradable polyester with a number of industrial applications that make it attractive as a potential replacement of oil-based plastics. The non-selective nature of chromosomal insertions of the biosynthetic genes was evidenced by a large landscape of PHB synthesis levels in independent clones. One clone was selected and further characterized as a microbial cell factory for PHB accumulation, and it achieved polymer accumulation levels comparable to those of a plasmid-bearing recombinant. Taken together, our results demonstrate that the new mini-Tn5 vectors can be used to confer interesting phenotypes in Gram-negative bacteria that would be very difficult to engineer through direct manipulation of the structural genes.

  2. Epigenetic changes and transposon reactivation in Thai rice hybrids. Molecular Breeding

    NARCIS (Netherlands)

    Kantama, L.; Junbuathong, S.; Sakulkoo, J.; Jong, de J.H.S.G.M.; Apisitwanich, S.

    2013-01-01

    Inter- or intraspecific hybridization is the first step in transferring exogenous traits to the germplasm of a recipient crop. One of the complicating factors is the occurrence of epigenetic modifications of the hybrids, which in turn can change their gene expression and phenotype. In this study we

  3. Negative effect of the 5'-untranslated leader sequence on Ac transposon promoter expression.

    Science.gov (United States)

    Scortecci, K C; Raina, R; Fedoroff, N V; Van Sluys, M A

    1999-08-01

    Transposable elements are used in heterologous plant hosts to clone genes by insertional mutagenesis. The Activator (Ac) transposable element has been cloned from maize, and introduced into a variety of plants. However, differences in regulation and transposition frequency have been observed between different host plants. The cause of this variability is still unknown. To better understand the activity of the Ac element, we analyzed the Ac promoter region and its 5'-untranslated leader sequence (5' UTL). Transient assays in tobacco NT1 suspension cells showed that the Ac promoter is a weak promoter and its activity was localized by deletion analyses. The data presented here indicate that the core of the Ac promoter is contained within 153 bp fragment upstream to transcription start sites. An important inhibitory effect (80%) due to the presence of the 5' UTL was found on the expression of LUC reporter gene. Here we demonstrate that the presence of the 5' UTL in the constructs reduces the expression driven by either strong or weak promoters.

  4. The W22 genome: a foundation for maize functional genomics and transposon biology

    Science.gov (United States)

    The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using small-read sequencing technologies. We show that significant structural heterogeneity exists in ...

  5. Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx.

    Science.gov (United States)

    Suárez, Gabriel A; Renda, Brian A; Dasgupta, Aurko; Barrick, Jeffrey E

    2017-09-01

    The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS 1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS 1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS 1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase ( dinP ) and a DNA damage response regulator ( umuD Ab [the umuD gene of A. baylyi ]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves, inserting into other genes, and thereby inactivating them from the ADP1 genome. The resulting "clean-genome" ADP1-ISx strain exhibited larger reductions in the rates of inactivating mutations than expected from spontaneous mutation rates measured via whole-genome sequencing of lineages evolved under relaxed selection. Surprisingly, we also found that IS element activity reduces transformability and is a major cause of cell aggregation and death in wild-type ADP1 grown under normal laboratory conditions. More generally, our results demonstrate that domesticating a bacterial genome by removing mobile DNA elements that have accumulated during evolution in the wild can have unanticipated benefits. Copyright © 2017 American Society for Microbiology.

  6. Noncoding origins of anthropoid traits and a new null model of transposon functionalization.

    Science.gov (United States)

    del Rosario, Ricardo C H; Rayan, Nirmala Arul; Prabhakar, Shyam

    2014-09-01

    Little is known about novel genetic elements that drove the emergence of anthropoid primates. We exploited the sequencing of the marmoset genome to identify 23,849 anthropoid-specific constrained (ASC) regions and confirmed their robust functional signatures. Of the ASC base pairs, 99.7% were noncoding, suggesting that novel anthropoid functional elements were overwhelmingly cis-regulatory. ASCs were highly enriched in loci associated with fetal brain development, motor coordination, neurotransmission, and vision, thus providing a large set of candidate elements for exploring the molecular basis of hallmark primate traits. We validated ASC192 as a primate-specific enhancer in proliferative zones of the developing brain. Unexpectedly, transposable elements (TEs) contributed to >56% of ASCs, and almost all TE families showed functional potential similar to that of nonrepetitive DNA. Three L1PA repeat-derived ASCs displayed coherent eye-enhancer function, thus demonstrating that the "gene-battery" model of TE functionalization applies to enhancers in vivo. Our study provides fundamental insights into genome evolution and the origins of anthropoid phenotypes and supports an elegantly simple new null model of TE exaptation. © 2014 del Rosario et al.; Published by Cold Spring Harbor Laboratory Press.

  7. Germline transgenesis in rodents by pronuclear microinjection of Sleeping Beauty transposons

    Czech Academy of Sciences Publication Activity Database

    Ivics, Z.; Mátés, L.; Yau, T. Y.; Landa, Vladimír; Zídek, Václav; Bashir, S.; Hoffmann, O. I.; Hiripi, L.; Garrels, W.; Kues, W. A.; Bösze, Z.; Geurts, A.; Pravenec, Michal; Rülicke, T.; Izsvák, Z.

    2014-01-01

    Roč. 9, č. 4 (2014), s. 773-793 ISSN 1754-2189 R&D Projects: GA TA ČR(CZ) TA02010013; GA MŠk(CZ) LH12061; GA MŠk(CZ) LL1204 Institutional support: RVO:67985823 Keywords : mouse * rat * transgenesis * Sleeping Beauty * protocol Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 9.673, year: 2014

  8. Transposon mutagenesis to improve the growth of recombinant Saccharomyces cerevisiae on D-xylose

    Science.gov (United States)

    Haiying Ni; Jose M. Laplaza; Thomas W. Jeffries

    2007-01-01

    Saccharomyces cerevisiae L2612 transformed with genes for xylose reductase and xylitol dehydrogenase (XYL1 and XYL2) grows well on glucose but very poorly on D-xylose. When a gene for D-xylulokinase (XYL3 or XKS1) is overexpressed, growth on glucose is unaffected, but growth on xylose is blocked. Spontaneous or chemically induced mutants of this engineered yeast that...

  9. Knockout of an outer membrane protein operon of anaplasma marginale by transposon mutagenesis

    Science.gov (United States)

    Large amounts of data generated by genomics, transcriptomics and proteomics technologies have increased our understanding of the biology of Anaplasma marginale. However, these data have also led to new assumptions that require testing, ideally through classic genetic mutation. One example is the def...

  10. Meiotic recombination breakpoints are associated with open chromatin and enriched with Stowaway transposons in potato

    Science.gov (United States)

    Meiotic recombination is the foundation for genetic variation in natural and artificial populations of eukaryotes. Although genetic recombination maps have been developed in numerous plant species since late the 1980s, very few of these maps have provided the necessary resolution needed to investiga...

  11. New Transposon Tools Tailored for Metabolic Engineering of Gram-Negative Microbial Cell Factories

    International Nuclear Information System (INIS)

    Martínez-García, Esteban; Aparicio, Tomás; Lorenzo, Víctor de; Nikel, Pablo I.

    2014-01-01

    Re-programming microorganisms to modify their existing functions and/or to bestow bacteria with entirely new-to-Nature tasks have largely relied so far on specialized molecular biology tools. Such endeavors are not only relevant in the burgeoning metabolic engineering arena but also instrumental to explore the functioning of complex regulatory networks from a fundamental point of view. À la carte modification of bacterial genomes thus calls for novel tools to make genetic manipulations easier. We propose the use of a series of new broad-host-range mini-Tn5-vectors, termed pBAMDs, for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for generating saturated mutagenesis libraries in gene function studies. These delivery vectors endow the user with the possibility of easy cloning and subsequent insertion of functional cargoes with three different antibiotic-resistance markers (kanamycin, streptomycin, and gentamicin). After validating the pBAMD vectors in the environmental bacterium Pseudomonas putida KT2440, their use was also illustrated by inserting the entire poly(3-hydroxybutyrate) (PHB) synthesis pathway from Cupriavidus necator in the chromosome of a phosphotransacetylase mutant of Escherichia coli. PHB is a completely biodegradable polyester with a number of industrial applications that make it attractive as a potential replacement of oil-based plastics. The non-selective nature of chromosomal insertions of the biosynthetic genes was evidenced by a large landscape of PHB synthesis levels in independent clones. One clone was selected and further characterized as a microbial cell factory for PHB accumulation, and it achieved polymer accumulation levels comparable to those of a plasmid-bearing recombinant. Taken together, our results demonstrate that the new mini-Tn5-vectors can be used to confer interesting phenotypes in Gram-negative bacteria that would be very difficult to engineer through direct manipulation of the structural genes.

  12. An Enterobacter Plasmid as a New Genetic Background for the Transposon Tn1331

    Science.gov (United States)

    2011-11-25

    determined to be 99% similar to E. cloacae by both 16S rDNA and Phoenix analysis and was designated Enterobacter sp W001. Enterobacter sp W001 was...adolescents. JAMA. 2002;287(23):3096–3102. 9. Foster TJ. Plasmid- determined resistance to antimicrobial drugs and toxic metal ions in bacteria. Microbiol...mediated type II dihydrofolate reductase gene among trimethoprim -resistant urinary pathogens in Greek hospitals. J Antimicrob Chemother. 1992;29

  13. New Transposon Tools Tailored for Metabolic Engineering of Gram-Negative Microbial Cell Factories

    Energy Technology Data Exchange (ETDEWEB)

    Martínez-García, Esteban; Aparicio, Tomás; Lorenzo, Víctor de; Nikel, Pablo I., E-mail: pablo.nikel@cnb.csic.es [Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Madrid (Spain)

    2014-10-28

    Re-programming microorganisms to modify their existing functions and/or to bestow bacteria with entirely new-to-Nature tasks have largely relied so far on specialized molecular biology tools. Such endeavors are not only relevant in the burgeoning metabolic engineering arena but also instrumental to explore the functioning of complex regulatory networks from a fundamental point of view. À la carte modification of bacterial genomes thus calls for novel tools to make genetic manipulations easier. We propose the use of a series of new broad-host-range mini-Tn5-vectors, termed pBAMDs, for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for generating saturated mutagenesis libraries in gene function studies. These delivery vectors endow the user with the possibility of easy cloning and subsequent insertion of functional cargoes with three different antibiotic-resistance markers (kanamycin, streptomycin, and gentamicin). After validating the pBAMD vectors in the environmental bacterium Pseudomonas putida KT2440, their use was also illustrated by inserting the entire poly(3-hydroxybutyrate) (PHB) synthesis pathway from Cupriavidus necator in the chromosome of a phosphotransacetylase mutant of Escherichia coli. PHB is a completely biodegradable polyester with a number of industrial applications that make it attractive as a potential replacement of oil-based plastics. The non-selective nature of chromosomal insertions of the biosynthetic genes was evidenced by a large landscape of PHB synthesis levels in independent clones. One clone was selected and further characterized as a microbial cell factory for PHB accumulation, and it achieved polymer accumulation levels comparable to those of a plasmid-bearing recombinant. Taken together, our results demonstrate that the new mini-Tn5-vectors can be used to confer interesting phenotypes in Gram-negative bacteria that would be very difficult to engineer through direct manipulation of the structural genes.

  14. Vast potential for using the piggyBac transposon to engineer transgenic plants

    Science.gov (United States)

    The acceptance of bioengineered plants by some nations is hampered by a number of factors, including the random insertion of a transgene into the host genome. Emerging technologies, such as site-specific nucleases, are enabling plant scientists to promote recombination or mutations at specific plant...

  15. Computational prediction and molecular confirmation of Helitron transposons in the maize genome

    Directory of Open Access Journals (Sweden)

    He Limei

    2008-01-01

    Full Text Available Abstract Background Helitrons represent a new class of transposable elements recently uncovered in plants and animals. One remarkable feature of Helitrons is their ability to capture gene sequences, which makes them of considerable potential evolutionary importance. However, because Helitrons lack the typical structural features of other DNA transposable elements, identifying them is a challenge. Currently, most researchers identify Helitrons manually by comparing sequences. With the maize whole genome sequencing project underway, an automated computational Helitron searching tool is needed. The characterization of Helitron activities in maize needs to be addressed in order to better understand the impact of Helitrons on the organization of the genome. Results We developed and implemented a heuristic searching algorithm in PERL for identifying Helitrons. Our HelitronFinder program will (i take FASTA-formatted DNA sequences as input and identify the hairpin looping patterns, and (ii exploit the consensus 5' and 3' end sequences of known Helitrons to identify putative ends. We randomly selected five predicted Helitrons from the program's high quality output for molecular verification. Four out of the five predicted Helitrons were confirmed by PCR assays and DNA sequencing in different maize inbred lines. The HelitronFinder program identified two head-to-head dissimilar Helitrons in a maize BAC sequence. Conclusion We have identified 140 new Helitron candidates in maize with our computational tool HelitronFinder by searching maize DNA sequences currently available in GenBank. Four out of five candidates were confirmed to be real by empirical methods, thus validating the predictions of HelitronFinder. Additional points to emerge from our study are that Helitrons do not always insert at an AT dinucleotide in the host sequences, that they can insert immediately adjacent to an existing Helitron, and that their movement may cause changes in the flanking region, such as deletions.

  16. Drosophila Transposon Insertions as Unknowns for Structured Inquiry Recombination Mapping Exercises in an Undergraduate Genetics Course

    OpenAIRE

    Marcus, Jeffrey M.; Hughes, Tia M.

    2009-01-01

    Structured inquiry approaches, in which students receive a Drosophila strain of unknown genotype to analyze and map the constituent mutations, are a common feature of many genetics teaching laboratories. The required crosses frustrate many students because they are aware that they are participating in a fundamentally trivial exercise, as the map locations of the genes are already established and have been recalculated thousands of times by generations of students. We modified the traditional ...

  17. Identification of flagellar motility genes in Yersinia ruckeri by transposon mutagenesis

    DEFF Research Database (Denmark)

    Evenhuis, Jason P:; LaPatra, Scott E.; Verner-Jeffreys, David W.

    2009-01-01

    Here we demonstrate that flagellar secretion is required for production of secreted lipase activity in the fish pathogen Yersinia ruckeri and that neither of these activities is necessary for virulence in rainbow trout. Our results suggest a possible mechanism for the emergence of nonmotile biotype...

  18. Microarray Analysis of Transposon Insertion Mutants in Bacillus Anthracis: Global Identification of Genes Required for Sporulation and Germination

    National Research Council Canada - National Science Library

    Day , Jr., William A; Rasmussen, Suzanne L; Carpenter, Beth M; Peterson, Scott N; Friedlander, Arthur M

    2007-01-01

    .... The system, used to identify genes required for generation of the infectious anthrax spore, spore germination and optimal growth on rich medium, was predictive of the contribution of two conserved...

  19. Arabidopsis RNA Polymerase V Mediates Enhanced Compaction and Silencing of Geminivirus and Transposon Chromatin during Host Recovery from Infection.

    Science.gov (United States)

    Coursey, Tami; Regedanz, Elizabeth; Bisaro, David M

    2018-04-01

    Plants employ RNA-directed DNA methylation (RdDM) and dimethylation of histone 3 lysine 9 (H3K9me2) to silence geminiviruses and transposable elements (TEs). We previously showed that canonical RdDM (Pol IV-RdDM) involving RNA polymerases IV and V (Pol IV and Pol V) is required for Arabidopsis thaliana to recover from infection with Beet curly top virus lacking a suppressor protein that inhibits methylation (BCTV L2 - ). Recovery, which is characterized by reduced viral DNA levels and symptom remission, allows normal floral development. Here, we used formaldehyde-assisted isolation of regulatory elements (FAIRE) to confirm that >90% of BCTV L2 - chromatin is highly compacted during recovery, and a micrococcal nuclease-chromatin immunoprecipitation assay showed that this is largely due to increased nucleosome occupancy. Physical compaction correlated with augmented cytosine and H3K9 methylation and with reduced viral gene expression. We additionally demonstrated that these phenomena are dependent on Pol V and by extension the Pol IV-RdDM pathway. BCTV L2 - was also used to evaluate the impact of viral infection on host loci, including repressed retrotransposons Ta3 and Athila6A Remarkably, an unexpected Pol V-dependent hypersuppression of these TEs was observed, resulting in transcript levels even lower than those detected in uninfected plants. Hypersuppression is likely to be especially important for natural recovery from wild-type geminiviruses, as viral L2 and AL2 proteins cause ectopic TE expression. Thus, Pol IV-RdDM targets both viral and TE chromatin during recovery, simultaneously silencing the majority of viral genomes and maintaining host genome integrity by enforcing tighter control of TEs in future reproductive tissues. IMPORTANCE In plants, RdDM pathways use small RNAs to target cytosine and H3K9 methylation, thereby silencing DNA virus genomes and transposable elements (TEs). Further, Pol IV-RdDM involving Pol IV and Pol V is a key aspect of host defense that can lead to recovery from geminivirus infection. Recovery is characterized by reduced viral DNA levels and symptom remission and thus allows normal floral development. Studies described here demonstrate that the Pol V-dependent enhanced viral DNA and histone methylation observed during recovery result in increased chromatin compaction and suppressed gene expression. In addition, we show that TE-associated chromatin is also targeted for hypersuppression during recovery, such that TE transcripts are reduced below the already low levels seen in uninfected plants. Thus, Pol IV-RdDM at once silences the majority of viral genomes and enforces a tight control over TEs which might otherwise jeopardize genome integrity in future reproductive tissue. Copyright © 2018 American Society for Microbiology.

  20. Genome-Wide Transposon Mutagenesis Indicates that Mycobacterium marinum Customizes Its Virulence Mechanisms for Survival and Replication in Different Hosts

    KAUST Repository

    Weerdenburg, Eveline M.; Abdallah, Abdallah; Rangkuti, Farania; Abd El Ghany, Moataz; Otto, Thomas D.; Adroub, Sabir; Molenaar, Douwe; Ummels, Roy; ter Veen, Kars; van Stempvoort, Gunny; van der Sar, Astrid M.; Ali, Shahjahan; Langridge, Gemma C.; Thomson, Nicholas R.; Pain, Arnab; Bitter, Wilbert

    2015-01-01

    the type VII protein secretion system ESX-1, biosynthesis of polyketide lipids, and utilization of sterols. However, we were also able to show that M. marinum contains an even larger set of host-specific virulence determinants, including proteins involved

  1. Survey Sequencing Reveals Elevated DNA Transposon Activity, Novel Elements, and Variation in Repetitive Landscapes among Vesper Bats

    Czech Academy of Sciences Publication Activity Database

    Pagán, H.J.T.; Macas, Jiří; Novák, Petr; McCulloch, E.S.; Stevens, R.D.; Ray, D.A.

    2012-01-01

    Roč. 4, č. 4 (2012), s. 575-585 ISSN 1759-6653 Institutional research plan: CEZ:AV0Z50510513 Keywords : HORIZONTAL TRANSFER * AUTOMATED CLASSIFICATION * GENOME SEQUENCE Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.759, year: 2012

  2. Successive increases in the resistance of Drosophila to viral infection through a transposon insertion followed by a Duplication.

    Directory of Open Access Journals (Sweden)

    Michael M Magwire

    2011-10-01

    Full Text Available To understand the molecular basis of how hosts evolve resistance to their parasites, we have investigated the genes that cause variation in the susceptibility of Drosophila melanogaster to viral infection. Using a host-specific pathogen of D. melanogaster called the sigma virus (Rhabdoviridae, we mapped a major-effect polymorphism to a region containing two paralogous genes called CHKov1 and CHKov2. In a panel of inbred fly lines, we found that a transposable element insertion in the protein coding sequence of CHKov1 is associated with increased resistance to infection. Previous research has shown that this insertion results in a truncated messenger RNA that encodes a far shorter protein than the susceptible allele. This resistant allele has rapidly increased in frequency under directional selection and is now the commonest form of the gene in natural populations. Using genetic mapping and site-specific recombination, we identified a third genotype with considerably greater resistance that is currently rare in the wild. In these flies there have been two duplications, resulting in three copies of both the truncated allele of CHKov1 and CHKov2 (one of which is also truncated. Remarkably, the truncated allele of CHKov1 has previously been found to confer resistance to organophosphate insecticides. As estimates of the age of this allele predate the use of insecticides, it is likely that this allele initially functioned as a defence against viruses and fortuitously "pre-adapted" flies to insecticides. These results demonstrate that strong selection by parasites for increased host resistance can result in major genetic changes and rapid shifts in allele frequencies; and, contrary to the prevailing view that resistance to pathogens can be a costly trait to evolve, the pleiotropic effects of these changes can have unexpected benefits.

  3. Transposon characterization of vancomycin-resistant Enterococcus faecium (VREF) and dissemination of resistance associated with transferable plasmids

    DEFF Research Database (Denmark)

    Migura, Lourdes Garcia; Liebana, Ernesto; Jensen, Lars Bogø

    2007-01-01

    Objectives: VanA glycopeptide resistance has persisted on broiler farms in the UK despite the absence of the antimicrobial selective pressure, avoparcin. This study aimed to investigate the contribution of horizontal gene transfer of Tn 1546 versus clonal spread in the dissemination of the resist......Objectives: VanA glycopeptide resistance has persisted on broiler farms in the UK despite the absence of the antimicrobial selective pressure, avoparcin. This study aimed to investigate the contribution of horizontal gene transfer of Tn 1546 versus clonal spread in the dissemination...... plasmid replicons, associated with antimicrobial resistance on several unrelated farms. Conclusions: Horizontal transfer of vancomycin resistance may play a more important role in the persistence of antimicrobial resistance than clonal spread. The presence of different plasmid replicons, associated...... with antimicrobial resistance on several unrelated farms, illustrates the ability of these enterococci to acquire and disseminate mobile genetic elements within integrated livestock systems....

  4. Toward the development of an efficient transposon marker system for soil bacteria based on the gusA gene

    International Nuclear Information System (INIS)

    Corbo, Joseph C.

    1991-01-01

    On account of the numerous drawbacks of presently existing marker gene systems, we have decided to concentrate on the use of the gusA gene as a marker, since it avoids many of the problems encountered by these other systems. Before I discuss the goals of my work, I would like briefly to describe the beta-gtucuronidase enzyme, its structural gene gusA, and their normal function in E. coli. The gusA gene was originally isolated from E. coli, one of the major constituents of the microflora of the mammalian gastrointestinal tract. In its native host it forms an operon with two other genes, gusB and gusC, and is involved in the uptake and degradation of glucuronidated compounds. Since glucuronidation (the covalent attachment of a glucuronide group) is the principal means of detoxification of xenobiotic compounds in human beings, it is reasonable enough that E. co//should have evolved such an enzyme. By selectively taking up the glucuronated compounds which it encounters in the gut, E. co//acquires a food source, glucuronic acid, which it obtains by hydrolyzing the glucuronide from its aglycone by means of the beta-glucuronidase enzyme. When we assay beta-glucuronidase activity we employ a colorless substrate known as X-gluc which has the chemical formula 5-bromo-4-chloro-3-indolyl beta-D-glucuronide. Upon removal of the glucuronide group the aglycone quickly dimerizes via oxidation to produce an insoluble, deep blue precipitate, which is readily identifiable as the hallmark sign of beta-glucuronidase activity. In this work it was tried to accomplish two related goals: the first goal was to build a marker gene construct using the gusA gene that would meet the requirements for a marker system that I have outlined above; the second goal was to find the best possible marker delivery system that would allow maximal ease of transfer of the marker gene from a host strain to the soil bacterium which is to be marked

  5. Phenotypic diversification is associated with host-induced transposon derepression in the sudden oak death pathogen Phytophthora ramorum

    Science.gov (United States)

    T. Kasuga; M. Kozanitas; M. Bui; D. Huberli; D. M. Rizzo; M. Garbelotto

    2012-01-01

    The oomycete pathogen Phytophthora ramorum is responsible for sudden oak death (SOD) in California coastal forests. P. ramorum is a generalist pathogen with over 100 known host species. Three or four closely related genotypes of P. ramorum (from a single lineage) were...

  6. A Sleeping Beauty DNA transposon-based genetic sensor for functional screening of vitamin D3 analogues

    DEFF Research Database (Denmark)

    Staunstrup, Nicklas Heine; Sharma, Nynne; Bak, Rasmus Otkjær

    2011-01-01

    Analogues of vitamin D3 are extensively used in the treatment of various illnesses, such as osteoporosis, inflammatory skin diseases, and cancer. Functional testing of new vitamin D3 analogues and formulations for improved systemic and topical administration is supported by sensitive screening me...

  7. A Noise Trimming and Positional Significance of Transposon Insertion System to Identify Essential Genes in Yersinia pestis

    Science.gov (United States)

    Yang, Zheng Rong; Bullifent, Helen L.; Moore, Karen; Paszkiewicz, Konrad; Saint, Richard J.; Southern, Stephanie J.; Champion, Olivia L.; Senior, Nicola J.; Sarkar-Tyson, Mitali; Oyston, Petra C. F.; Atkins, Timothy P.; Titball, Richard W.

    2017-02-01

    Massively parallel sequencing technology coupled with saturation mutagenesis has provided new and global insights into gene functions and roles. At a simplistic level, the frequency of mutations within genes can indicate the degree of essentiality. However, this approach neglects to take account of the positional significance of mutations - the function of a gene is less likely to be disrupted by a mutation close to the distal ends. Therefore, a systematic bioinformatics approach to improve the reliability of essential gene identification is desirable. We report here a parametric model which introduces a novel mutation feature together with a noise trimming approach to predict the biological significance of Tn5 mutations. We show improved performance of essential gene prediction in the bacterium Yersinia pestis, the causative agent of plague. This method would have broad applicability to other organisms and to the identification of genes which are essential for competitiveness or survival under a broad range of stresses.

  8. Development of CACTA transposon derived SCAR markers and their use in population structure analysis in Zea mays.

    Science.gov (United States)

    Roy, Neha Samir; Park, Kyong-Cheul; Lee, Sung-Il; Im, Min-Ji; Ramekar, Rahul Vasudeo; Kim, Nam-Soo

    2018-02-01

    Molecular marker technologies have proven to be an important breakthrough for genetic studies, construction of linkage maps and population genetics analysis. Transposable elements (TEs) constitute major fractions of repetitive sequences in plants and offer a wide range of possible areas to be explored as molecular markers. Sequence characterized amplified region (SCAR) marker development provides us with a simple and time saving alternative approach for marker development. We employed the CACTA-TD to develop SCARs and then integrated them into linkage map and used them for population structure and genetic diversity analysis of corn inbred population. A total of 108 dominant SCAR markers were designed out of which, 32 were successfully integrated in to the linkage map of maize RIL population and the remaining were added to a physical map for references to check the distribution throughout all chromosomes. Moreover, 76 polymorphic SCARs were used for diversity analysis of corn accessions being used in Korean corn breeding program. The overall average polymorphic information content (PIC) was 0.34, expected heterozygosity was 0.324 and Shannon's information index was 0.491 with a percentage of polymorphism of 98.67%. Further analysis by associating with desirable traits may also provide some accurate trait specific tagged SCAR markers. TE linked SCARs can provide an added level of polymorphism as well as improved discriminating ability and therefore can be useful in further breeding programs to develop high yielding germplasm.

  9. A transposon-directed epigenetic change in ZmCCT underlies quantitative resistance to Gibberella stalk rot in maize.

    Science.gov (United States)

    Wang, Chao; Yang, Qin; Wang, Weixiang; Li, Yipu; Guo, Yanling; Zhang, Dongfeng; Ma, Xuena; Song, Wei; Zhao, Jiuran; Xu, Mingliang

    2017-09-01

    A major resistance quantitative trait locus, qRfg1, significantly enhances maize resistance to Gibberella stalk rot, a devastating disease caused by Fusarium graminearum. However, the underlying molecular mechanism remains unknown. We adopted a map-based cloning approach to identify the resistance gene at qRfg1 and examined the dynamic epigenetic changes during qRfg1-mediated maize resistance to the disease. A CCT domain-containing gene, ZmCCT, is the causal gene at the qRfg1 locus and a polymorphic CACTA-like transposable element (TE1) c. 2.4 kb upstream of ZmCCT is the genetic determinant of allelic variation. The non-TE1 ZmCCT allele is in a poised state, with predictive bivalent chromatin enriched for both repressive (H3K27me3/H3K9me3) and active (H3K4me3) histone marks. Upon pathogen challenge, this non-TE1 ZmCCT allele was promptly induced by a rapid yet transient reduction in H3K27me3/H3K9me3 and a progressive decrease in H3K4me3, leading to disease resistance. However, TE1 insertion in ZmCCT caused selective depletion of H3K4me3 and enrichment of methylated GC to suppress the pathogen-induced ZmCCT expression, resulting in disease susceptibility. Moreover, ZmCCT-mediated resistance to Gibberella stalk rot is not affected by photoperiod sensitivity. This chromatin-based regulatory mechanism enables ZmCCT to be more precise and timely in defense against F. graminearum infection. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  10. Molecular genetics and epigenetics of CACTA elements

    KAUST Repository

    Fedoroff, Nina V.

    2013-01-01

    The CACTA transposons, so named for a highly conserved motif at element ends, comprise one of the most abundant superfamilies of Class 2 (cut-and-paste) plant transposons. CACTA transposons characteristically include subterminal sequences of several

  11. Transposon mutagenesis of Salmonella Enteritidis identifies genes that contribute to invasiveness in human and chicken cells and survival in egg albumen.

    Science.gov (United States)

    Salmonella Enteritidis is the world’s leading cause of food borne salmonellosis and illness in people is linked strongly to its contamination of eggs produced by otherwise healthy appearing hens. Salmonella Enteritidis is noted for generating exceptional strain heterogeneity despite having a clonal ...

  12. Genome sequencing and transposon mutagenesis of Burkholderia seminalis TC3.4.2R3 identify genes contributing to suppression of orchid necrosis caused by B. gladioli

    Science.gov (United States)

    Thirty six strains of Burkholderia spp. isolated from sugarcane were evaluated for biological control of leaf and pseudobulb necrosis of orchid caused by B. gladioli. Twenty nine of the sugarcane strains suppressed the disease in greenhouse assays. We generated a draft genomic sequence of one suppr...

  13. Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries

    DEFF Research Database (Denmark)

    Becker, F.; Schnorr, K.; Wilting, R.

    2004-01-01

    To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less beta-lactamase ('bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The 'bla gene was cloned into a bacteriophage MU...... minitransposon enabling translational fusions between 'bla and target genes. Fusion of TnSig in the correct reading frame to a protein carrying transmembrane domains or signal peptides resulted in ampicillin resistance of the corresponding clone. Prokaryotic gene libraries from the alkaliphilic bacterium...... Bacillus halodurans C125 and the hyperthermophilic archaeon Sulfolobus solfataricus P2 were tagged with TnSig. The genomic sequences, which are publicly available (EMBL BA000004 and EMBL AE006641), were used for rapid open reading frame (ORF) identification and prediction of protein localisation...

  14. Two Different Tetracycline Resistance Mechanisms, Plasmid-Carried tet(L) and Chromosomally Located Transposon-Associated tet(M), Coexist in Lactobacillus sakei Rits 9

    NARCIS (Netherlands)

    Ammor, M.S.; Gueimonde, M.; Danielsen, M.; Zagorec, M.; Hoek, van A.H.A.M.; Reyes-Gavilán, de los C.G.; Mayo, B.; Margolles, A.

    2008-01-01

    Lactobacillus sakei is extensively used as functional starter culture in fermented meat products. One of the safety criteria of a starter culture is the absence of potentially transferable antibiotic resistance determinants. However, tetracycline-resistant L. sakei strains have already been

  15. Shifts in the evolutionary rate and intensity of purifying selection between two Brassica genomes revealed by analyses of orthologous transposons and relics of a whole genome triplication.

    Science.gov (United States)

    Zhao, Meixia; Du, Jianchang; Lin, Feng; Tong, Chaobo; Yu, Jingyin; Huang, Shunmou; Wang, Xiaowu; Liu, Shengyi; Ma, Jianxin

    2013-10-01

    Recent sequencing of the Brassica rapa and Brassica oleracea genomes revealed extremely contrasting genomic features such as the abundance and distribution of transposable elements between the two genomes. However, whether and how these structural differentiations may have influenced the evolutionary rates of the two genomes since their split from a common ancestor are unknown. Here, we investigated and compared the rates of nucleotide substitution between two long terminal repeats (LTRs) of individual orthologous LTR-retrotransposons, the rates of synonymous and non-synonymous substitution among triplicated genes retained in both genomes from a shared whole genome triplication event, and the rates of genetic recombination estimated/deduced by the comparison of physical and genetic distances along chromosomes and ratios of solo LTRs to intact elements. Overall, LTR sequences and genic sequences showed more rapid nucleotide substitution in B. rapa than in B. oleracea. Synonymous substitution of triplicated genes retained from a shared whole genome triplication was detected at higher rates in B. rapa than in B. oleracea. Interestingly, non-synonymous substitution was observed at lower rates in the former than in the latter, indicating shifted densities of purifying selection between the two genomes. In addition to evolutionary asymmetry, orthologous genes differentially regulated and/or disrupted by transposable elements between the two genomes were also characterized. Our analyses suggest that local genomic and epigenomic features, such as recombination rates and chromatin dynamics reshaped by independent proliferation of transposable elements and elimination between the two genomes, are perhaps partially the causes and partially the outcomes of the observed inter-specific asymmetric evolution. © 2013 Purdue University The Plant Journal © 2013 John Wiley & Sons Ltd.

  16. First detection of the staphylococcal trimethoprim resistance gene dfrK and the dfrK-carrying transposon Tn559 in enterococci.

    Science.gov (United States)

    López, María; Kadlec, Kristina; Schwarz, Stefan; Torres, Carmen

    2012-02-01

    The trimethoprim resistance gene dfrK has been recently described in Staphylococcus aureus, but so far has not been found in other bacteria. A total of 166 enterococci of different species (E. faecium, E. faecalis, E. hirae, E. durans, E. gallinarum, and E. casseliflavus) and origins (food, clinical diseases in humans, healthy humans or animals, and sewage) were studied for their susceptibility to trimethoprim as determined by agar dilution (European Committee on Antimicrobial Susceptibility Testing) and the presence of (a) the dfrK gene and its genetic environment and (b) other dfr genes. The dfrK gene was detected in 49% of the enterococci (64% and 42% of isolates with minimum inhibitory concentrations of ≥2 mg/L or ≤1 mg/L, respectively). The tet(L)-dfrK linkage was detected in 21% of dfrK-positive enterococci. The chromosomal location of the dfrK gene was identified in one E. faecium isolate in which the dfrK was not linked to tet(L) gene but was part of a Tn559 element, which was integrated in the chromosomal radC gene. This Tn559 element was also found in 14 additional isolates. All combinations of dfr genes were detected among the isolates tested (dfrK, dfrG, dfrF, dfrK+dfrG, dfrK+dfrF, dfrF+dfrG, and dfrF+dfrG+dfrK). The gene dfrK gene was found together with other dfr genes in 58% of the tested enterococci. This study suggested an exchange of the trimethoprim resistance gene dfrK between enterococci and staphylococci, as previously observed for the trimethoprim resistance gene dfrG.

  17. High-Level Heat Resistance of Spores of Bacillus amyloliquefaciens and Bacillus licheniformis Results from the Presence of a spoVA Operon in a Tn1546 Transposon

    NARCIS (Netherlands)

    Berendsen, Erwin M; Koning, Rosella A; Boekhorst, Jos; de Jong, Anne; Kuipers, Oscar P; Wells-Bennik, Marjon H J

    2016-01-01

    Bacterial endospore formers can produce spores that are resistant to many food processing conditions, including heat. Some spores may survive heating processes aimed at production of commercially sterile foods. Recently, it was shown that a spoVA operon, designated spoVA(2mob), present on a Tn1546

  18. A transposon mutant library of Bacillus cereus ATCC 10987 reveals novel genes required for biofilm formation and implicates motility as an important factor for pellicle-biofilm formation

    DEFF Research Database (Denmark)

    Okshevsky, Mira; Louw, Matilde Greve; Lamela, Elena Otero

    2018-01-01

    Bacillus cereus is one of the most common opportunistic pathogens causing foodborne illness, as well as a common source of contamination in the dairy industry. B. cereus can form robust biofilms on food processing surfaces, resulting in food contamination due to shedding of cells and spores. Desp...

  19. The identification of a tetracycline resistance gene tet(M), on a Tn916-like transposon, in the Bacillus cereus group

    DEFF Research Database (Denmark)

    Agersø, Yvonne; Jensen, Lars Bogø; Givskov, Michael Christian

    2002-01-01

    In order to investigate whether resistance genes present in bacteria in manure could transfer to indigenous soil bacteria, resistant isolates belonging to the Bacillus cereus group (Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis) were isolated from farm soil (72 isolates) and manure...

  20. SCREEN FOR DOMINANT BEHAVIORAL MUTATIONS CAUSED BY GENOMIC INSERTION OF P-ELEMENT TRANSPOSONS IN DROSOPHILA: AN EXAMINATION OF THE INTEGRATION OF VIRAL VECTOR SEQUENCES

    OpenAIRE

    FOX, LYLE E.; GREEN, DAVID; YAN, ZIYING; ENGELHARDT, JOHN F.; WU, CHUN-FANG

    2007-01-01

    Here we report the development of a high-throughput screen to assess dominant mutation rates caused by P-element transposition within the Drosophila genome that is suitable for assessing the undesirable effects of integrating foreign regulatory sequences (viral cargo) into a host genome. Three different behavioral paradigms were used: sensitivity to mechanical stress, response to heat stress, and ability to fly. The results, from our screen of 35,000 flies, indicate that mutations caused by t...

  1. Global alteration of microRNAs and transposon-derived small RNAs in cotton (Gossypium hirsutum) during Cotton leafroll dwarf polerovirus (CLRDV) infection.

    Science.gov (United States)

    Romanel, Elisson; Silva, Tatiane F; Corrêa, Régis L; Farinelli, Laurent; Hawkins, Jennifer S; Schrago, Carlos E G; Vaslin, Maite F S

    2012-11-01

    Small RNAs (sRNAs) are a class of non-coding RNAs ranging from 20- to 40-nucleotides (nts) that are present in most eukaryotic organisms. In plants, sRNAs are involved in the regulation of development, the maintenance of genome stability and the antiviral response. Viruses, however, can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of sRNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). To understand the impact of viral infection on the plant sRNA pathway, we deep sequenced the sRNAs in cotton leaves infected with Cotton leafroll dwarf virus (CLRDV), which is a member of the economically important virus family Luteoviridae. A total of 60 putative conserved cotton miRNAs were identified, including 19 new miRNA families that had not been previously described in cotton. Some of these miRNAs were clearly misregulated during viral infection, and their possible role in symptom development and disease progression is discussed. Furthermore, we found that the 24-nt heterochromatin-associated siRNAs were quantitatively and qualitatively altered in the infected plant, leading to the reactivation of at least one cotton transposable element. This is the first study to explore the global alterations of sRNAs in virus-infected cotton plants. Our results indicate that some CLRDV-induced symptoms may be correlated with the deregulation of miRNA and/or epigenetic networks.

  2. Revised genomic consensus for the hypermethylated CpG island region of the human L1 transposon and integration sites of full length L1 elements from recombinant clones made using methylation-tolerant host strains

    DEFF Research Database (Denmark)

    Crowther, P J; Doherty, J P; Linsenmeyer, M E

    1991-01-01

    preferentially from L1 members which have accumulated mutations that have removed sites of methylation. We present a revised consensus from the 5' presumptive control region of these elements. This revised consensus contains a consensus RNA polymerase III promoter which would permit the synthesis of transcripts......Efficient recovery of clones from the 5' end of the human L1 dispersed repetitive elements necessitates the use of deletion mcr- host strains since this region contains a CpG island which is hypermethylated in vivo. Clones recovered with conventional mcr+ hosts seem to have been derived...... from the 5' end of full length L1 elements. Such potential transcripts are likely to exhibit a high degree of secondary structure. In addition, we have determined the flanking sequences for 6 full length L1 elements. The majority of full length L1 clones show no convincing evidence for target site...

  3. A transposon mutant library of Bacillus cereus ATCC 10987 reveals novel genes required for biofilm formation and implicates motility as an important factor for pellicle-biofilm formation

    DEFF Research Database (Denmark)

    Okshevsky, Mira Ursula; Louw, Matilde Greve; Otero Lamela, Elena

    2018-01-01

    Bacillus cereus is one of the most common opportunistic pathogens causing foodborne illness, as well as a common source of contamination in the dairy industry. B. cereus can form robust biofilms on food processing surfaces, resulting in food contamination due to shedding of cells and spores. Desp...

  4. Cytosine methylation does not affect binding of transcription factor Sp1

    International Nuclear Information System (INIS)

    Harrington, M.A.; Jones, P.A.; Imagawa, M.; Karin, M.

    1988-01-01

    DNA methylation may be a component of a multilevel control mechanism that regulates eukaryotic gene expression. The authors used synthetic oligonucleotides to investigate the effect of cytosine methylation on the binding of the transcription factor Sp1 to its target sequence (a G+C-rich sequence known as a GC box). Concatemers of double-stranded 14-mers containing a GC box successfully competed with the human metallothionein IIA promoter for binding to Sp1 in DNase I protection experiments. The presence of 5-methylcytosine in the CpG sequence of the GC box did not influence Sp1 binding. The result was confirmed using double-stranded 20-mers containing 16 base pairs of complementary sequence. Electrophoretic gel retardation analysis of annealed 28-mers containing a GC box incubated with an Sp1-containing HeLa cell nuclear extract demonstrated the formation of DNA-protein complexes; formation of these complexes was not inhibited when an oligomer without a GC box was used as a competitor. Once again, the presence of a 5-methylcytosine residue in the GC box did not influence the binding of the protein to DNA. The results therefore preclude a direct effect of cytosine methylation on Sp1-DNA interactions

  5. SERDP CU-1129 Biological Assessment for Characterizing Contamination Risk at the Genetic-, Individual-, and Population-Level

    Science.gov (United States)

    2004-04-14

    albimanus Quetzal : a Tc1 transposon (Sum P(2) = 8.3e-07) Transposase_5, E = 5.9e-08 Topo 6-2L3 LpTc1.1 A. albimanus Quetzal : a Tc1 transposon (Sum P(2...transposon LpTc1, A. albimanus Quetzal = Anopheles albimanus Quetzal transposase (AAB02109). The clones tank and tank.1 did not have sufficient

  6. The biology and potential for genetic research of transposable elements in filamentous fungi

    OpenAIRE

    Fávaro,Léia Cecilia de Lima; Araújo,Welington Luiz de; Azevedo,João Lúcio de; Paccola-Meirelles,Luzia Doretto

    2005-01-01

    Recently many transposable elements have been identified and characterized in filamentous fungi, especially in species of agricultural, biotechnological and medical interest. Similar to the elements found in other eukaryotes, fungal transposons can be classified as class I elements (retrotransposons) that use RNA and reverse transcriptase and class II elements (DNA transposons) that use DNA. The changes (transposition and recombination) caused by transposons can supply wide-ranging genetic va...

  7. Genomic analysis of 495 vancomycin-resistant Enterococcus faecium reveals broad dissemination of a vanA plasmid in more than 19 clones from Copenhagen, Denmark

    DEFF Research Database (Denmark)

    Pinholt, Mette; Gumpert, Heidi; Bayliss, Sion

    2017-01-01

    . The majority of the isolates were located in three groups (n = 44, 100 and 218) and clonal spread of VREfm between wards and hospitals was identified. Five Tn1546-like transposon types were identified. A dominant truncated transposon (type 4, 92%) was spread across all but one VREfm group. The closed van......A plasmid was highly covered by reads from isolates containing the type 4 transposon. CONCLUSIONS: This study suggests that it was the dissemination of the type 4 Tn1546-like transposon and plasmid via horizontal transfer to multiple populations of E. faecium, followed by clonal spread of new VREfm clones...

  8. Mutations in the Histone-Like Nucleoid Structuring Regulatory Gene (hns) Decrease the Adherence of Shiga Toxin-Producing Escherichia coli 091:H21 Strain B2F1 to Human Colonic Epithelial Cells and Increase the Production of Hemolysin

    National Research Council Canada - National Science Library

    Scott, Maria

    1999-01-01

    ...:H21 to human colonic epithelial cells. Transposon mutagenesis of B2F1 was accomplished with the mini-Tn5phoACm mobile element and a mutant bank of B2F1 colonies that carried putative in-frame PhoA-positive transposon insertions was isolated...

  9. Sudha Bhattacharya Jawaharlal Nehru University New Delhi

    Indian Academy of Sciences (India)

    TSC

    Bottom strand. 1. 2. 3. G A T C. 5. 1. 3. 4. 2. *. Nicking profile of EN on the 174 bp substrate. Insertion point. Bottom strand labelled 174bp substrate. Transposon insertion point. Endonuclease nicks at three hot spots. The transposon inserts at hot spot # 3 ...

  10. GenBank blastx search result: AK242295 [KOME

    Lifescience Database Archive (English)

    Full Text Available e cds; transposons Ophelia2, Angela3s, and XJ3, complete sequence; LRR protein WM1.3 (WM1.3) gene, complete cds; Stowaway... MITE, transposons XJ1, Jody, Angela, and XJ and Stowaway MITE, comp

  11. GenBank blastx search result: AK241352 [KOME

    Lifescience Database Archive (English)

    Full Text Available e cds; transposons Ophelia2, Angela3s, and XJ3, complete sequence; LRR protein WM1.3 (WM1.3) gene, complete cds; Stowaway... MITE, transposons XJ1, Jody, Angela, and XJ and Stowaway MITE, comp

  12. GenBank blastn search result: AK241152 [KOME

    Lifescience Database Archive (English)

    Full Text Available e cds; transposons Ophelia2, Angela3s, and XJ3, complete sequence; LRR protein WM1.3 (WM1.3) gene, complete cds; Stowaway... MITE, transposons XJ1, Jody, Angela, and XJ and Stowaway MITE, comp

  13. GenBank blastx search result: AK241749 [KOME

    Lifescience Database Archive (English)

    Full Text Available e cds; transposons Ophelia2, Angela3s, and XJ3, complete sequence; LRR protein WM1.3 (WM1.3) gene, complete cds; Stowaway... MITE, transposons XJ1, Jody, Angela, and XJ and Stowaway MITE, comp

  14. GenBank blastx search result: AK241418 [KOME

    Lifescience Database Archive (English)

    Full Text Available e cds; transposons Ophelia2, Angela3s, and XJ3, complete sequence; LRR protein WM1.3 (WM1.3) gene, complete cds; Stowaway... MITE, transposons XJ1, Jody, Angela, and XJ and Stowaway MITE, comp

  15. GenBank blastx search result: AK241200 [KOME

    Lifescience Database Archive (English)

    Full Text Available e cds; transposons Ophelia2, Angela3s, and XJ3, complete sequence; LRR protein WM1.3 (WM1.3) gene, complete cds; Stowaway... MITE, transposons XJ1, Jody, Angela, and XJ and Stowaway MITE, comp

  16. GenBank blastx search result: AK240882 [KOME

    Lifescience Database Archive (English)

    Full Text Available e cds; transposons Ophelia2, Angela3s, and XJ3, complete sequence; LRR protein WM1.3 (WM1.3) gene, complete cds; Stowaway... MITE, transposons XJ1, Jody, Angela, and XJ and Stowaway MITE, comp

  17. Calcium chloride improve ethanol production in recombinant ...

    African Journals Online (AJOL)

    The T7-expression system has been very useful for protein expression in Escherichia coli. Here, a T7- expression transposon was constructed, which allowed simple construction of T7-expression Zymomonas mobilis. This transposon contained the T7 RNA polymerase being driven by the gap promoter from Z. mobilis.

  18. ORF Alignment: NC_003888 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_003888 gi|21218662 >1cxqA 5 133 141 282 2e-06 ... ref|NP_624441.1| putative noncomposite... transposon transposase [Streptomyces ... coelicolor A3(2)] emb|CAB52910.1| putative noncomposite... ... transposon transposase [Streptomyces coelicolor A3(2)] ... pir||T36996 probable noncomposite

  19. Analysis of a MULE-cyanide hydratase gene fusion in Verticillium dahliae

    Science.gov (United States)

    The genome of the phytopathogenic fungus Verticillium dahliae encodes numerous Class II “cut-and-paste” transposable elements, including those of a small group of MULE transposons. We have previously identified a fusion event between a MULE transposon sequence and sequence encoding a cyanide hydrata...

  20. A Small RNA-Based Immune System Defends Germ Cells against Mobile Genetic Elements

    Directory of Open Access Journals (Sweden)

    Astrid D. Haase

    2016-01-01

    Full Text Available Transposons are mobile genetic elements that threaten the survival of species by destabilizing the germline genomes. Limiting the spread of these selfish elements is imperative. Germ cells employ specialized small regulatory RNA pathways to restrain transposon activity. PIWI proteins and Piwi-interacting RNAs (piRNAs silence transposons at the transcriptional and posttranscriptional level with loss-of-function mutant animals universally exhibiting sterility often associated with germ cell defects. This short review aims to illustrate basic strategies of piRNA-guided defense against transposons. Mechanisms of piRNA silencing are most readily studied in Drosophila melanogaster, which serves as a model to delineate molecular concepts and as a reference for mammalian piRNA systems. PiRNA pathways utilize two major strategies to handle the challenges of transposon control: (1 the hard-wired molecular memory of prior transpositions enables recognition of mobile genetic elements and discriminates transposons from host genes; (2 a feed-forward adaptation mechanism shapes piRNA populations to selectively combat the immediate threat of transposon transcripts. In flies, maternally contributed PIWI-piRNA complexes bolster both of these lines of defense and ensure transgenerational immunity. While recent studies have provided a conceptual framework of what could be viewed as an ancient immune system, we are just beginning to appreciate its many molecular innovations.

  1. Assessing Tn5 and Sleeping Beauty for transpositional transgenesis by cytoplasmic injection into bovine and ovine zygotes

    Science.gov (United States)

    Bevacqua, R. J.; Fernandez-Martin, R.; Canel, N. G.; Gibbons, A.; Texeira, D.; Lange, F.; Vans Landschoot, G.; Savy, V.; Briski, O.; Hiriart, M. I.; Grueso, E.; Ivics, Z.; Taboga, O.; Kues, W. A.; Ferraris, S.

    2017-01-01

    Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes. In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes, employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest. PMID:28301581

  2. Expression of the gene cluster associated with the Escherichia coli pilus adhesin K99.

    OpenAIRE

    Lee, J H; Isaacson, R E

    1995-01-01

    The biogenesis of the pilus adhesin K99 is dependent on the expression of eight contiguous genes, fanA to fanH. Transposon mutants were prepared by using TnlacZ and TnphoA, and selected transposon mutants were used to measure expression of each K99 gene. Expression of the K99 genes is likely controlled at the transcription level, since in general, there were no differences between the results obtained with the two transposons. fanC was the most highly expressed, and fanD was expressed at very...

  3. Comparing insertion libraries in two Pseudomonas aeruginosa strains to assess gene essentiality.

    Science.gov (United States)

    Liberati, Nicole T; Urbach, Jonathan M; Thurber, Tara K; Wu, Gang; Ausubel, Frederick M

    2008-01-01

    Putative essential genes can be identified by comparing orthologs not disrupted in multiple near-saturated transposon insertion mutation libraries in related strains of the same bacterial species. Methods for identifying all orthologs between two bacterial strains and putative essential orthologs are described. In addition, protocols detailing near-saturation transposon insertion mutagenesis of bacteria are presented, including (1) conjugation-mediated mutagenesis, (2) automated colony picking and liquid handling of mutant cultures, and (3) arbitrary polymerase chain reaction amplification and sequencing of genomic DNA adjacent to transposon insertion sites.

  4. The biology and potential for genetic research of transposable elements in filamentous fungi

    Directory of Open Access Journals (Sweden)

    Léia Cecilia de Lima Fávaro

    2005-12-01

    Full Text Available Recently many transposable elements have been identified and characterized in filamentous fungi, especially in species of agricultural, biotechnological and medical interest. Similar to the elements found in other eukaryotes, fungal transposons can be classified as class I elements (retrotransposons that use RNA and reverse transcriptase and class II elements (DNA transposons that use DNA. The changes (transposition and recombination caused by transposons can supply wide-ranging genetic variation, especially for species that do not have a sexual phase. The application of transposable elements to gene isolation and population analysis is an important tool for molecular biology and studies of fungal evolution.

  5. Antibiotics and Resistance: Glossary

    Science.gov (United States)

    ... chromosomes and plasmids. Transposons often carry genes specifying antimicrobial resistance. Virus An extremely small infective agent, visible only with an electron microscope. Viruses can cause disease in humans, animals and plants. Viruses consist of a protein coat ...

  6. Piwi-interacting RNAs (piRNAs) in animals: The story so far

    African Journals Online (AJOL)

    STORAGESEVER

    2009-09-01

    Sep 1, 2009 ... germline stem cell maintenance, epigenetic regulation, and transposition. Although the ... Species. MILI. (PIWIL2 In human. Transposon, and piRNA clusters. 24-28 nts .... However, the differences among embryonic, neonatal,.

  7. Fulltext PDF

    Indian Academy of Sciences (India)

    Unknown

    A four-element based transposon system for allele specific tagging in plants ... 191. BRCA1. Analysis of BRCA1 involvement in breast cancer in Indian women. 19. Brain ... Cortical tissue. Transfer of learning across the somatosensory cortex. 5.

  8. Fulltext PDF

    Indian Academy of Sciences (India)

    Prakash

    2010-05-04

    May 4, 2010 ... circumstances due to 'buffering' activity of Hsp90. A recent study ... this is a mobilization of transposons – a process by which these ... of this phenomenon with new studies pointing to antibiotic-induced mutagenesis in bacteria.

  9. Proteomics technique opens new frontiers in mobilome research

    OpenAIRE

    Davidson, Andrew; Matthews, David; Maringer, Kevin

    2017-01-01

    ABSTRACT A large proportion of the genome of most eukaryotic organisms consists of highly repetitive mobile genetic elements. The sum of these elements is called the ?mobilome,? which in eukaryotes is made up mostly of transposons. Transposable elements contribute to disease, evolution, and normal physiology by mediating genetic rearrangement, and through the ?domestication? of transposon proteins for cellular functions. Although ?omics studies of mobilome genomes and transcriptomes are commo...

  10. Genetic Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and Isolation of a Colorless Mutant

    OpenAIRE

    Van Dien, Stephen J.; Marx, Christopher J.; O'Brien, Brooke N.; Lidstrom, Mary E.

    2003-01-01

    Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, r...

  11. Annotation and sequence diversity of transposable elements in common bean (Phaseolus vulgaris

    Directory of Open Access Journals (Sweden)

    Scott eJackson

    2014-07-01

    Full Text Available Common bean (Phaseolus vulgaris is an important legume crop grown and consumed worldwide. With the availability of the common bean genome sequence, the next challenge is to annotate the genome and characterize functional DNA elements. Transposable elements (TEs are the most abundant component of plant genomes and can dramatically affect genome evolution and genetic variation. Thus, it is pivotal to identify TEs in the common bean genome. In this study, we performed a genome-wide transposon annotation in common bean using a combination of homology and sequence structure-based methods. We developed a 2.12-Mb transposon database which includes 791 representative transposon sequences and is available upon request or from www.phytozome.org. Of note, nearly all transposons in the database are previously unrecognized TEs. More than 5,000 transposon-related expressed sequence tags (ESTs were detected which indicates that some transposons may be transcriptionally active. Two Ty1-copia retrotransposon families were found to encode the envelope-like protein which has rarely been identified in plant genomes. Also, we identified an extra open reading frame (ORF termed ORF2 from 15 Ty3-gypsy families that was located between the ORF encoding the retrotransposase and the 3’LTR. The ORF2 was in opposite transcriptional orientation to retrotransposase. Sequence homology searches and phylogenetic analysis suggested that the ORF2 may have an ancient origin, but its function is not clear. This transposon data provides a useful resource for understanding the genome organization and evolution and may be used to identify active TEs for developing transposon-tagging system in common bean and other related genomes.

  12. Cold Spring Harbor symposia on quantitative biology: Volume 49, Recombination at the DNA level

    International Nuclear Information System (INIS)

    1984-01-01

    This volume contains full papers prepared by the participants to the 1984 Cold Springs Harbor Symposia on Quantitative Biology. This year's theme is entitled Recombination at the DNA level. The volume consists of 93 articles grouped into subject areas entitled chromosome mechanics, yeast systems, mammalian homologous recombination, transposons, mu, plant transposons/T4 recombination, topoisomerase, resolvase and gyrase, Escherichia coli general recombination, RecA, repair, leukaryotic enzymes, integration and excision of bacteriophage, site-specific recombination, and recombination in vitro

  13. ORF Alignment: NC_003384 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available [Zymomonas mobilis] gb|AAQ67239.1| ... Bla [Transposon delivery vector pUT-miniTn5-gfp-tet] ... ... ... 3.5.2.6) precursor - Escherichia coli plasmids ... gb|AAC79082.1| beta lactamase [Transposon delivery...AG47772.1| beta-lactamase ... TEM-1 [Klebsiella pneumoniae] gb|AAG37887.1| ampicillin ... resistance [Tn10 delivery... vector pHV1249] gb|AAG37882.1| ... ampicillin resistance [Tn10 delivery

  14. The Genome of the Basidiomycetous Yeast and Human Pathogen Cryptococcus neoformans

    OpenAIRE

    Loftus, Brendan J.; Fung, Eula; Roncaglia, Paola; Rowley, Don; Amedeo, Paolo; Bruno, Dan; Vamathevan, Jessica; Miranda, Molly; Anderson, Iain J.; Fraser, James A.; Allen, Jonathan E.; Bosdet, Ian E.; Brent, Michael R.; Chiu, Readman; Doering, Tamara L.

    2005-01-01

    Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its ~20-megabase genome, which contains ~6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype i...

  15. Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

    OpenAIRE

    Metts, J; West, J; Doares, S H; Matthysse, A G

    1991-01-01

    Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was d...

  16. Comprehensive identification of Salmonella enterica serovar typhimurium genes required for infection of BALB/c mice.

    Directory of Open Access Journals (Sweden)

    Roy R Chaudhuri

    2009-07-01

    Full Text Available Genes required for infection of mice by Salmonella Typhimurium can be identified by the interrogation of random transposon mutant libraries for mutants that cannot survive in vivo. Inactivation of such genes produces attenuated S. Typhimurium strains that have potential for use as live attenuated vaccines. A quantitative screen, Transposon Mediated Differential Hybridisation (TMDH, has been developed that identifies those members of a large library of transposon mutants that are attenuated. TMDH employs custom transposons with outward-facing T7 and SP6 promoters. Fluorescently-labelled transcripts from the promoters are hybridised to whole-genome tiling microarrays, to allow the position of the transposon insertions to be determined. Comparison of microarray data from the mutant library grown in vitro (input with equivalent data produced after passage of the library through mice (output enables an attenuation score to be determined for each transposon mutant. These scores are significantly correlated with bacterial counts obtained during infection of mice using mutants with individual defined deletions of the same genes. Defined deletion mutants of several novel targets identified in the TMDH screen are effective live vaccines.

  17. Molecular genetics and epigenetics of CACTA elements

    KAUST Repository

    Fedoroff, Nina V.

    2013-08-21

    The CACTA transposons, so named for a highly conserved motif at element ends, comprise one of the most abundant superfamilies of Class 2 (cut-and-paste) plant transposons. CACTA transposons characteristically include subterminal sequences of several hundred nucleotides containing closely spaced direct and inverted repeats of a short, conserved sequence of 14-15 bp. The Supressor-mutator (Spm) transposon, identified and subjected to detailed genetic analysis by Barbara McClintock, remains the paradigmatic element of the CACTA family. The Spm transposon encodes two proteins required for transposition, the transposase (TnpD) and a regulatory protein (TnpA) that binds to the subterminal repeats. Spm expression is subject to both genetic and epigenetic regulation. The Spm-encoded TnpA serves as an activator of the epigenetically inactivated, methylated Spm, stimulating both transient and heritable activation of the transposon. TnpA also serves as a negative regulator of the demethylated active element promoter and is required, in addition to the TnpD, for transposition. © Springer Science+Business Media, New York 2013.

  18. Transposable elements are enriched within or in close proximity to xenobiotic-metabolizing cytochrome P450 genes

    Directory of Open Access Journals (Sweden)

    Li Xianchun

    2007-03-01

    Full Text Available Abstract Background Transposons, i.e. transposable elements (TEs, are the major internal spontaneous mutation agents for the variability of eukaryotic genomes. To address the general issue of whether transposons mediate genomic changes in environment-adaptation genes, we scanned two alleles per each of the six xenobiotic-metabolizing Helicoverpa zea cytochrome P450 loci, including CYP6B8, CYP6B27, CYP321A1, CYP321A2, CYP9A12v3 and CYP9A14, for the presence of transposon insertions by genome walking and sequence analysis. We also scanned thirteen Drosophila melanogaster P450s genes for TE insertions by in silico mapping and literature search. Results Twelve novel transposons, including LINEs (long interspersed nuclear elements, SINEs (short interspersed nuclear elements, MITEs (miniature inverted-repeat transposable elements, one full-length transib-like transposon, and one full-length Tcl-like DNA transpson, are identified from the alleles of the six H. zea P450 genes. The twelve transposons are inserted into the 5'flanking region, 3'flanking region, exon, or intron of the six environment-adaptation P450 genes. In D. melanogaster, seven out of the eight Drosophila P450s (CYP4E2, CYP6A2, CYP6A8, CYP6A9, CYP6G1, CYP6W1, CYP12A4, CYP12D1 implicated in insecticide resistance are associated with a variety of transposons. By contrast, all the five Drosophila P450s (CYP302A1, CYP306A1, CYP307A1, CYP314A1 and CYP315A1 involved in ecdysone biosynthesis and developmental regulation are free of TE insertions. Conclusion These results indicate that TEs are selectively retained within or in close proximity to xenobiotic-metabolizing P450 genes.

  19. Diversity of the Tetracycline Mobilome within a Chinese Pig Manure Sample.

    Science.gov (United States)

    Leclercq, Sébastien Olivier; Wang, Chao; Zhu, Yaxin; Wu, Hai; Du, Xiaochen; Liu, Zhipei; Feng, Jie

    2016-11-01

    Tetracycline antibiotics are widely used in livestock, and tetracycline resistance genes (TRG) are frequently reported in the manure of farmed animals. However, the diversity of TRG-carrying transposons in manure has still been rarely investigated. Using a culture-free functional metagenomic procedure, combined with large-insert library construction and sequencing, bioinformatic analyses, and functional experiments, we identified 17 distinct TRGs in a single pig manure sample, including two new tet genes: tet(59), encoding a tetracycline efflux pump, and tet(W/N/W), encoding mosaic ribosomal protection. Our study also revealed six new TRG-carrying putative nonconjugative transposons: Tn5706-like transposon Tn6298, IS200/605-related transposon Tn6303, Tn3 family transposon Tn6299, and three ISCR2-related transposons, Tn62300, Tn62301, and Tn62302 IMPORTANCE: Fertilization of agricultural fields with animal manure is believed to play a major role in antibiotic resistance dissemination in the environment. There is growing concern for the possible spread of antibiotic resistance from the environment to humans since genetic resistance determinants may be located in transposons and other mobile genetic elements potentially transferable to pathogens. Among the various antibiotic resistance genes found in manure, tetracycline resistance genes (TRGs) are some of the most common. The present study provides a detailed snapshot of the tetracycline mobilome in a single pig manure sample, revealing an unappreciated diversity of TRGs and potential TRG mobility vectors. Our precise identification of the TRG-carrying units will enable us to investigate in more details their mobility effectiveness. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  20. A Solution to the C-Value Paradox and the Function of Junk DNA: The Genome Balance Hypothesis.

    Science.gov (United States)

    Freeling, Michael; Xu, Jie; Woodhouse, Margaret; Lisch, Damon

    2015-06-01

    The Genome Balance Hypothesis originated from a recent study that provided a mechanism for the phenomenon of genome dominance in ancient polyploids: unique 24nt RNA coverage near genes is greater in genes on the recessive subgenome irrespective of differences in gene expression. 24nt RNAs target transposons. Transposon position effects are now hypothesized to balance the expression of networked genes and provide spring-like tension between pericentromeric heterochromatin and microtubules. The balance (coordination) of gene expression and centromere movement is under selection. Our hypothesis states that this balance can be maintained by many or few transposons about equally well. We explain known balanced distributions of junk DNA within genomes and between subgenomes in allopolyploids (and our hypothesis passes "the onion test" for any so-called solution to the C-value paradox). Importantly, when the allotetraploid maize chromosomes delete redundant genes, their nearby transposons are also lost; this result is explained if transposons near genes function. The Genome Balance Hypothesis is hypothetical because the position effect mechanisms implicated are not proved to apply to all junk DNA, and the continuous nature of the centromeric and gene position effects have not yet been studied as a single phenomenon. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  1. Cooperation is fleeting in the world of transposable elements.

    Directory of Open Access Journals (Sweden)

    Andreas Wagner

    2006-12-01

    Full Text Available Composite transposons are key vehicles for the worldwide spreading of genes that allow bacteria to survive toxic compounds. Composite transposons consist of two smaller transposable elements called insertion sequences (ISs, which flank the genes that permit such survival. Each IS in a composite transposon can either transpose alone, selfishly, or it can transpose cooperatively, jointly with the other IS. Cooperative transposition can enhance an IS's chance of survival, but it also carries the risk of transposon destruction. I use game theory to show that the conditions under which cooperative transposition is an evolutionarily stable strategy (ESS are not biologically realistic. I then analyze the distribution of thousands of ISs in more than 200 bacterial genomes to test the following prediction of the game-theoretical model: if cooperative transposition was an ESS, then the closely spaced ISs that characterize composite transposons should be more abundant in genomes than expected by chance. The data show that this is not the case. Cooperativity can only be maintained in a transitional, far-from-equilibrium state shortly after a selection pressure first arises. This is the case in the spreading of antibiotic resistance, where we are witnessing a fleeting moment in evolution, a moment in which cooperation among selfish DNA molecules has provided a means of survival. Because such cooperation does not pay in the long run, the vehicles of such survival will eventually disappear again. My analysis demonstrates that game theory can help explain behavioral strategies even for mobile DNA.

  2. Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

    Directory of Open Access Journals (Sweden)

    Nynne Sharma

    2013-01-01

    Full Text Available DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system.

  3. One to rule them all

    Science.gov (United States)

    Bouuaert, Corentin Claeys; Tellier, Michael; Chalmers, Ronald

    2014-01-01

    The development of transposon-based genome manipulation tools can benefit greatly from understanding transposons’ inherent regulatory mechanisms. The Tc1-mariner transposons, which are being widely used in biotechnological applications, are subject to a self-inhibitory mechanism whereby increasing transposase expression beyond a certain point decreases the rate of transposition. In a recent paper, Liu and Chalmers performed saturating mutagenesis on the highly conserved WVPHEL motif in the mariner-family transposase from the Hsmar1 element. Curiously, they found that the majority of all possible single mutations were hyperactive. Biochemical characterizations of the mutants revealed that the hyperactivity is due to a defect in communication between transposase subunits, which normally regulates transposition by reducing the rate of synapsis. This provides important clues for improving transposon-based tools. However, some WVPHEL mutants also showed features that would be undesirable for most biotechnological applications: they showed uncontrolled DNA cleavage activities and defects in the coordination of cleavage between the two transposon ends. The study illustrates how the knowledge of inhibitory mechanisms can help improve transposon tools but also highlights an important challenge, which is to specifically target a regulatory mechanism without affecting other important functions of the transposase. PMID:24812590

  4. Characterization of the Complete Nucleotide Sequences of IncA/C2 Plasmids Carrying In809-Like Integrons from Enterobacteriaceae Isolates of Wildlife Origin.

    Science.gov (United States)

    Papagiannitsis, Costas C; Kutilova, Iva; Medvecky, Matej; Hrabak, Jaroslav; Dolejska, Monika

    2017-09-01

    A total of 18 Enterobacteriaceae (17 from gulls and 1 from a clinical sample) collected from Australia, carrying IncA/C plasmids with the IMP-encoding In809-like integrons, were studied. Seven plasmids, being representatives of different origins, plasmid sizes, replicon combinations, and resistance genes, were completely sequenced. Plasmid pEc158, identified in a clinical Escherichia coli ST752 isolate, showed extensive similarity to type 2 IncA/C 2 plasmids. pEc158 carried none of the bla CMY-2 -like region or ARI-B and ARI-A regions, while it contained a hybrid transposon structure. The six remaining plasmids, which were of wildlife origin, were highly similar to each other and probably were fusion derivatives of type 1 and type 2 A/C 2 plasmids. The latter plasmids contained an ARI-B region and hybrid transposon structures. In all plasmids, hybrid transposon structures containing In809-like integrons were inserted 3,434 bp downstream of the rhs2 start codon. In all cases, the one outermost 38-bp inverted repeat (IR) of the transposon was associated with the Tn 1696 tnp module, while the other outermost 38-bp IR of the transposon was associated with either a Tn 6317 -like module or a Tn 21 mer module. However, the internal structure of the transposon and the resistance genes were different in each plasmid. These findings indicated that, for the specific periods of time and settings, different IncA/C 2 plasmid types carrying In809-like elements circulated among isolates of wildlife and clinical origins. Additionally, they provided the basis for speculations regarding the reshuffling of IncA/C 2 plasmids with In809-like integrons and confirmed the rapid evolution of IncA/C 2 plasmid lineages. Copyright © 2017 American Society for Microbiology.

  5. Genetic Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and Isolation of a Colorless Mutant

    Science.gov (United States)

    Van Dien, Stephen J.; Marx, Christopher J.; O'Brien, Brooke N.; Lidstrom, Mary E.

    2003-01-01

    Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments. PMID:14660416

  6. Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

    DEFF Research Database (Denmark)

    Jakobsen, Jannik E; Li, Juan; Moldt, Brian

    2011-01-01

    We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon-based do......We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon...

  7. Genetic characterization of the carotenoid biosynthetic pathway in Methylobacterium extorquens AM1 and isolation of a colorless mutant.

    Science.gov (United States)

    Van Dien, Stephen J; Marx, Christopher J; O'Brien, Brooke N; Lidstrom, Mary E

    2003-12-01

    Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments.

  8. The Genome of the Basidiomycetous Yeast and Human Pathogen Cryptococcus neoformans

    Science.gov (United States)

    Loftus, Brendan J.; Fung, Eula; Roncaglia, Paola; Rowley, Don; Amedeo, Paolo; Bruno, Dan; Vamathevan, Jessica; Miranda, Molly; Anderson, Iain J.; Fraser, James A.; Allen, Jonathan E.; Bosdet, Ian E.; Brent, Michael R.; Chiu, Readman; Doering, Tamara L.; Donlin, Maureen J.; D’Souza, Cletus A.; Fox, Deborah S.; Grinberg, Viktoriya; Fu, Jianmin; Fukushima, Marilyn; Haas, Brian J.; Huang, James C.; Janbon, Guilhem; Jones, Steven J. M.; Koo, Hean L.; Krzywinski, Martin I.; Kwon-Chung, June K.; Lengeler, Klaus B.; Maiti, Rama; Marra, Marco A.; Marra, Robert E.; Mathewson, Carrie A.; Mitchell, Thomas G.; Pertea, Mihaela; Riggs, Florenta R.; Salzberg, Steven L.; Schein, Jacqueline E.; Shvartsbeyn, Alla; Shin, Heesun; Shumway, Martin; Specht, Charles A.; Suh, Bernard B.; Tenney, Aaron; Utterback, Terry R.; Wickes, Brian L.; Wortman, Jennifer R.; Wye, Natasja H.; Kronstad, James W.; Lodge, Jennifer K.; Heitman, Joseph; Davis, Ronald W.; Fraser, Claire M.; Hyman, Richard W.

    2012-01-01

    Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its ~20-megabase genome, which contains ~6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively spliced and antisense messages. The genome is rich in transposons, many of which cluster at candidate centromeric regions. The presence of these transposons may drive karyotype instability and phenotypic variation. C. neoformans encodes unique genes that may contribute to its unusual virulence properties, and comparison of two phenotypically distinct strains reveals variation in gene content in addition to sequence polymorphisms between the genomes. PMID:15653466

  9. Escherichia coli can be transformed by a liposome-mediated lipofection method.

    Science.gov (United States)

    Kawata, Yoshikazu; Yano, Shin-ichi; Kojima, Hiroyuki

    2003-05-01

    Transformation of Escherichia coli is a basic technique for genetic engineering. We used a liposome-mediated lipofection method to transform electrocompetent E. coli cells which has little natural competence of foreign DNA without electroporation treatment, and got transformants with simple and quick treatment by a plasmid or a transposon and transposase complex.

  10. RNA interference: its use as antiviral therapy

    NARCIS (Netherlands)

    Haasnoot, J.; Berkhout, B.

    2006-01-01

    RNA interference (RNAi) is a sequence-specific gene-silencing mechanism that has been proposed to function as a defence mechanism of eukaryotic cells against viruses and transposons. RNAi was first observed in plants in the form of a mysterious immune response to viral pathogens. But RNAi is more

  11. Activity of genes with functions in human Williams-Beuren Syndrome are impacted by mobile element insertions in the gray wolf genome.

    Science.gov (United States)

    vonHoldt, Bridgett M; Ji, Sarah S; Aardema, Matthew L; Stahler, Daniel; Udell, Monique A R; Sinsheimer, Janet S

    2018-06-01

    In canines, transposon dynamics have been associated with a hyper-social behavioral syndrome, although the functional mechanism has yet to be described. We investigate the epigenetic and transcriptional consequences of these behavior-associated mobile element insertions in dogs and Yellowstone wolves. We posit that the transposons themselves may not be the causative feature; rather, their transcriptional regulation may exert the functional impact. We survey four outlier transposons associated with hyper-sociability, with the expectation that they are targeted for epigenetic silencing. We predict hyper-methylation of mobile element insertions (MEIs), suggestive that the epigenetic silencing of and not the MEIs themselves may be driving dysregulation of nearby genes. We found that transposon-derived sequences are significantly hyper-methylated, regardless of their copy number or species. Further, we have assessed transcriptome sequence data and found evidence that mobile element insertions impact the expression levels of six genes (WBSCR17, LIMK1, GTF2I, WBSCR27, BAZ1B, and BCL7B), all of which have known roles in human Williams-Beuren syndrome due to changes in copy number, typically hemizygosity. Although further evidence is needed, our results suggest that a few insertions alter local expression at multiple genes, likely through a cis-regulatory mechanism that excludes proximal methylation.

  12. Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps

    Science.gov (United States)

    Andrew T. Groover; Joseph R. Fontana; Juana M. Arroyo; Cristina Yordan; W. Richard McCombie; Robert A. Martienssen

    2003-01-01

    Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a ß-glucuronidase reporter enzyme...

  13. UniProt search blastx result: AK289070 [KOME

    Lifescience Database Archive (English)

    Full Text Available ol3) (Transposon Ty3-2 TYA-TYB polyprotein) [Contains: Capsid protein (CA) (p24); Spacer peptide p3; Nucleoc...apsid protein p11 (NC); Ty3 protease (EC 3.4.23.-) (PR) (p16); Spacer peptide J; Reverse transcriptase/ribon

  14. UniProt search blastx result: AK288702 [KOME

    Lifescience Database Archive (English)

    Full Text Available ol3) (Transposon Ty3-1 TYA-TYB polyprotein) [Contains: Capsid protein (CA) (p24); Spacer peptide p3; Nucleoc...apsid protein p11 (NC); Ty3 protease (EC 3.4.23.-) (PR) (p16); Spacer peptide J; Reverse transcriptase/ribon

  15. UniProt search blastx result: AK289070 [KOME

    Lifescience Database Archive (English)

    Full Text Available ol3) (Transposon Ty3-1 TYA-TYB polyprotein) [Contains: Capsid protein (CA) (p24); Spacer peptide p3; Nucleoc...apsid protein p11 (NC); Ty3 protease (EC 3.4.23.-) (PR) (p16); Spacer peptide J; Reverse transcriptase/ribon

  16. Complete Sequence of p07-406, a 24,179-base-pair plasmid harboring the blaVIM-7 metallo-beta-lactamase gene in a Pseudomonas aeruginosa isolate from the United States.

    Science.gov (United States)

    Li, Hongyang; Toleman, Mark A; Bennett, Peter M; Jones, Ronald N; Walsh, Timothy R

    2008-09-01

    An outbreak involving a Pseudomonas aeruginosa strain that was resistant to all tested antimicrobials except polymyxin B occurred in a hospital in Houston, TX. Previous studies on this strain showed that it possesses a novel mobile metallo-beta-lactamase (MBL) gene, designated bla(VIM-7), located on a plasmid (p07-406). Here, we report the complete sequence, annotation, and functional characterization of this plasmid. p07-406 is 24,179 bp in length, and 29 open reading frames were identified related to known or putatively recognized proteins. Analysis of this plasmid showed it to be comprised of four distinct regions: (i) a region of 5,200 bp having a Tn501-like mercuric resistance (mer) transposon upstream of the replication region; (ii) a Tn3-like transposon carrying a truncated integron with a bla(VIM-7) gene and an insertion sequence inserted at the other end of this transposon; (iii) a region of four genes, upstream of the Tn3-like transposon, possessing very high similarity to plasmid pXcB from Xanthomonas campestris pv. citri commonly associated with plants; (iv) a backbone sequence similar to the backbone structure of the IncP group plasmid Rms149, pB10, and R751. This is the first plasmid to be sequenced carrying an MBL gene and highlights the amelioration of DNA segments from disparate origins, most noticeably from plant pathogens.

  17. Metabolic engineering to expand the substrate spectrum of Pseudomonas putida toward sucrose.

    Science.gov (United States)

    Löwe, Hannes; Schmauder, Lukas; Hobmeier, Karina; Kremling, Andreas; Pflüger-Grau, Katharina

    2017-08-01

    Sucrose is an important disaccharide used as a substrate in many industrial applications. It is a major component of molasses, a cheap by-product of the sugar industry. Unfortunately, not all industrially relevant organisms, among them Pseudomonas putida, are capable of metabolizing sucrose. We chose a metabolic engineering approach to circumvent this blockage and equip P. putida with the activities necessary to consume sucrose. Therefore, we constructed a pair of broad-host range mini-transposons (pSST - sucrose splitting transposon), carrying either cscA, encoding an invertase able to split sucrose into glucose and fructose, or additionally cscB, encoding a sucrose permease. Introduction of cscA was sufficient to convey sucrose consumption and the additional presence of cscB had no further effect, though the sucrose permease was built and localized to the membrane. Sucrose was split extracellularly by the activity of the invertase CscA leaking out of the cell. The transposons were also used to confer sucrose consumption to Cupriavidus necator. Interestingly, in this strain, CscB acted as a glucose transporter, such that C. necator also gained the ability to grow on glucose. Thus, the pSST transposons are functional tools to extend the substrate spectrum of Gram-negative bacterial strains toward sucrose. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  18. Dynamics of a Novel Highly Repetitive CACTA Family in Common Bean (Phaseolus vulgaris

    Directory of Open Access Journals (Sweden)

    Dongying Gao

    2016-07-01

    Full Text Available Transposons are ubiquitous genomic components that play pivotal roles in plant gene and genome evolution. We analyzed two genome sequences of common bean (Phaseolus vulgaris and identified a new CACTA transposon family named pvCACTA1. The family is extremely abundant, as more than 12,000 pvCACTA1 elements were found. To our knowledge, this is the most abundant CACTA family reported thus far. The computational and fluorescence in situ hybridization (FISH analyses indicated that the pvCACTA1 elements were concentrated in terminal regions of chromosomes and frequently generated AT-rich 3 bp target site duplications (TSD, WWW, W is A or T. Comparative analysis of the common bean genomes from two domesticated genetic pools revealed that new insertions or excisions of pvCACTA1 elements occurred after the divergence of the two common beans, and some of the polymorphic elements likely resulted in variation in gene sequences. pvCACTA1 elements were detected in related species but not outside the Phaseolus genus. We calculated the molecular evolutionary rate of pvCACTA1 transposons using orthologous elements that indicated that most transposition events likely occurred before the divergence of the two gene pools. These results reveal unique features and evolution of this new transposon family in the common bean genome.

  19. The use of hygromycin phosphotransferase gene (hpt) with an ...

    African Journals Online (AJOL)

    Previously, we have developed a marker-off system to truncate a selec marker in transgenic plants by locating one end of the transposon in the intron of the marker gene (glyphosate-tolerant epsps gene). In order to expand this technique to those marker genes without intron in this report, we created an artificial intron ...

  20. Blue ghosts: a new method for isolating amber mutants defective in essential genes of Escherichia coli

    DEFF Research Database (Denmark)

    Brown, S; Brickman, E R; Beckwith, J

    1981-01-01

    We describe a technique which permits an easy screening for amber mutants defective in essential genes of Escherichia coli. Using this approach, we have isolated three amber mutants defective in the rho gene. An extension of the technique allows the detection of ochre mutants and transposon inser...

  1. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    The hsrω gene is expressed widely at all developmental stages; in later embryonic stages, its expression in the developing central nervous system was prominent. In spite of insertion of a big transposon in the promoter, expression of the hsrω05241 allele in the enhancer-trap line, as revealed by in situ hybridization to hsrω ...

  2. The application of the high throughput sequencing technology in the transposable elements.

    Science.gov (United States)

    Liu, Zhen; Xu, Jian-hong

    2015-09-01

    High throughput sequencing technology has dramatically improved the efficiency of DNA sequencing, and decreased the costs to a great extent. Meanwhile, this technology usually has advantages of better specificity, higher sensitivity and accuracy. Therefore, it has been applied to the research on genetic variations, transcriptomics and epigenomics. Recently, this technology has been widely employed in the studies of transposable elements and has achieved fruitful results. In this review, we summarize the application of high throughput sequencing technology in the fields of transposable elements, including the estimation of transposon content, preference of target sites and distribution, insertion polymorphism and population frequency, identification of rare copies, transposon horizontal transfers as well as transposon tagging. We also briefly introduce the major common sequencing strategies and algorithms, their advantages and disadvantages, and the corresponding solutions. Finally, we envision the developing trends of high throughput sequencing technology, especially the third generation sequencing technology, and its application in transposon studies in the future, hopefully providing a comprehensive understanding and reference for related scientific researchers.

  3. Oryza sativa L.

    African Journals Online (AJOL)

    Jane

    2011-07-11

    Jul 11, 2011 ... Disease resistance in plants is a desirable economic trait. Many disease ... in BRGA-1(blast resistant gene analogue-1), putative retro-elements and putative retro-transposons proteins in ... with approximately 150 described in the Colombia ecotype of .... molecular basis of NBS2-Pi9 in broad-spectrum blast.

  4. A PiggyBac-mediated approach for muscle gene transfer or cell therapy

    Directory of Open Access Journals (Sweden)

    Déborah Ley

    2014-11-01

    Full Text Available An emerging therapeutic approach for Duchenne muscular dystrophy is the transplantation of autologous myogenic progenitor cells genetically modified to express dystrophin. The use of this approach is challenged by the difficulty in maintaining these cells ex vivo while keeping their myogenic potential, and ensuring sufficient transgene expression following their transplantation and myogenic differentiation in vivo. We investigated the use of the piggyBac transposon system to achieve stable gene expression when transferred to cultured mesoangioblasts and into murine muscles. Without selection, up to 8% of the mesoangioblasts expressed the transgene from 1 to 2 genomic copies of the piggyBac vector. Integration occurred mostly in intergenic genomic DNA and transgene expression was stable in vitro. Intramuscular transplantation of mouse Tibialis anterior muscles with mesoangioblasts containing the transposon led to sustained myofiber GFP expression in vivo. In contrast, the direct electroporation of the transposon-donor plasmids in the mouse Tibialis muscles in vivo did not lead to sustained transgene expression despite molecular evidence of piggyBac transposition in vivo. Together these findings provide a proof-of-principle that piggyBac transposon may be considered for mesoangioblast cell-based therapies of muscular dystrophies.

  5. Inhibition of virus replication by RNA interference

    NARCIS (Netherlands)

    Haasnoot, P. C. Joost; Cupac, Daniel; Berkhout, Ben

    2003-01-01

    RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene

  6. Biology Today symposium | Mid Year Meetings | Events | Indian ...

    Indian Academy of Sciences (India)

    11.15, D. P. KASBEKAR, Centre for Cellular and Molecular Biology, Hyderabad Neurospora abhors a transposon. 11.45, IMRAN SIDDIQI, Centre for Cellular and Molecular Biology, Hyderabad Meiotic chromosome organization. 12.15, VIDYANAND NANJUNDIAH, Indian Institute of Science, Bengaluru Social amoebae.

  7. pGLO Mutagenesis: A Laboratory Procedure in Molecular Biology for Biology Students

    Science.gov (United States)

    Bassiri, Eby A.

    2011-01-01

    A five-session laboratory project was designed to familiarize or increase the laboratory proficiency of biology students and others with techniques and instruments commonly used in molecular biology research laboratories and industries. In this project, the EZ-Tn5 transposon is used to generate and screen a large number of cells transformed with…

  8. The potential of virus-induced gene silencing for speeding up functional characterization of plant genes

    NARCIS (Netherlands)

    Benedito, V.A.; Visser, P.B.; Angenent, G.C.; Krens, F.A.

    2004-01-01

    Virus-induced gene silencing (VIGS) has been shown to be of great potential in plant reverse genetics. Advantages of VIGS over other approaches, such as T-DNA or transposon tagging, include the circumvention of plant transformation, methodological simplicity and robustness, and speedy results. These

  9. Genome-wide Screening Identifies Phosphotransferase System Permease BepA to Be Involved in Enterococcus faecium Endocarditis and Biofilm Formation

    NARCIS (Netherlands)

    Paganelli, Fernanda L.; Huebner, Johannes; Singh, Kavindra V.; Zhang, Xinglin; Van Schaik, Willem; Wobser, Dominique; Braat, Johanna C.; Murray, Barbara E.; Bonten, Marc J M; Willems, Rob J L; Leavis, Helen L.

    2016-01-01

    Enterococcus faecium is a common cause of nosocomial infections, of which infective endocarditis is associated with substantial mortality. In this study, we used a microarray-based transposon mapping (M-TraM) approach to evaluate a rat endocarditis model and identified a gene, originally annotated

  10. The piRNAs forge an immune system for the genome

    DEFF Research Database (Denmark)

    Muller, Sébastian; Pandey, R.R.; Pillai, R.S.

    2013-01-01

    Genome integrity of germline is essential for the survival of any species. A dedicated defence mechanism based on small RNA called piRNA (PIWI-interacting RNA) has evolved to protect the germline from the deleterious effects of transposon mobility in genomes such as mutations, deletions or chromo...

  11. DsRed gene expression by doxycycline in porcine fibroblasts and ...

    African Journals Online (AJOL)

    DsRed gene expression by doxycycline in porcine fibroblasts and cloned embryos using transposon. SuJin Kim, JoonHo Moon, BegoRoibas da Torre, Islam M Saadeldin, JungTaek Kang, JiYei Choi, SolJi Park, Byeong-Chun Lee, Goo Jang Goo Jang ...

  12. Comparative analysis of chimeric ZFP-, TALE- and Cas9-piggyBac transposases for integration into a single locus in human cells.

    Science.gov (United States)

    Luo, Wentian; Galvan, Daniel L; Woodard, Lauren E; Dorset, Dan; Levy, Shawn; Wilson, Matthew H

    2017-08-21

    Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

  13. RodZ and PgsA Play Intertwined Roles in Membrane Homeostasis of Bacillus subtilis and Resistance to Weak Organic Acid Stress

    NARCIS (Netherlands)

    van Beilen, Johan; Blohmke, Christoph J.; Folkerts, Hendrik; de Boer, Richard; Zakrzewska, Anna; Kulik, Wim; Vaz, Fred M.; Brul, Stanley; Ter Beek, Alexander

    2016-01-01

    Weak organic acids like sorbic and acetic acid are widely used to prevent growth of spoilage organisms such as Bacilli. To identify genes involved in weak acid stress tolerance we screened a transposon mutant library of Bacillus subtilis for sorbic acid sensitivity. Mutants of the rodZ (ymfM) gene

  14. An En/Spm based transposable element system for gene isolation in Arabidopsis thaliana

    NARCIS (Netherlands)

    Aarts, M.G.M.

    1996-01-01


    At the start of the research described in this thesis, the main aim was to develop, study and apply an efficient En/Spm-I/dSpm based transposon tagging system in Arabidopsis thaliana to generate tagged mutants and to provide insights in the

  15. A Graph Based Framework to Model Virus Integration Sites

    Directory of Open Access Journals (Sweden)

    Raffaele Fronza

    2016-01-01

    Here, we addressed the challenge to: 1 define the notion of CIS on graph models, 2 demonstrate that the structure of CIS enters in the category of scale-free networks and 3 show that our network approach analyzes CIS dynamically in an integrated systems biology framework using the Retroviral Transposon Tagged Cancer Gene Database (RTCGD as a testing dataset.

  16. Gene Discovery and Functional Analyses in the Model Plant Arabidopsis

    DEFF Research Database (Denmark)

    Feng, Cai-ping; Mundy, J.

    2006-01-01

    The present mini-review describes newer methods and strategies, including transposon and T-DNA insertions, TILLING, Deleteagene, and RNA interference, to functionally analyze genes of interest in the model plant Arabidopsis. The relative advantages and disadvantages of the systems are also discus...

  17. 40 CFR 725.239 - Use of specific microorganisms in activities conducted outside a structure.

    Science.gov (United States)

    2010-07-01

    ... Bradyrhizobium japonicum. (2) Modification of traits. (i) The introduced genetic material must meet the criteria for poorly mobilizable listed in § 725.421(c). (ii) The introduced genetic material must consist only... sequences needed to move genetic material, including linkers, homopolymers, adaptors, transposons, insertion...

  18. Germline transgenic pigs by Sleeping Beauty transposition in porcine zygotes and targeted integration in the pig genome.

    Directory of Open Access Journals (Sweden)

    Wiebke Garrels

    Full Text Available Genetic engineering can expand the utility of pigs for modeling human diseases, and for developing advanced therapeutic approaches. However, the inefficient production of transgenic pigs represents a technological bottleneck. Here, we assessed the hyperactive Sleeping Beauty (SB100X transposon system for enzyme-catalyzed transgene integration into the embryonic porcine genome. The components of the transposon vector system were microinjected as circular plasmids into the cytoplasm of porcine zygotes, resulting in high frequencies of transgenic fetuses and piglets. The transgenic animals showed normal development and persistent reporter gene expression for >12 months. Molecular hallmarks of transposition were confirmed by analysis of 25 genomic insertion sites. We demonstrate germ-line transmission, segregation of individual transposons, and continued, copy number-dependent transgene expression in F1-offspring. In addition, we demonstrate target-selected gene insertion into transposon-tagged genomic loci by Cre-loxP-based cassette exchange in somatic cells followed by nuclear transfer. Transposase-catalyzed transgenesis in a large mammalian species expands the arsenal of transgenic technologies for use in domestic animals and will facilitate the development of large animal models for human diseases.

  19. The cfr and cfr-like multiple resistance genes

    DEFF Research Database (Denmark)

    Vester, Birte

    2018-01-01

    . The cfr gene is found in various bacteria in many geographical locations and placed on plasmids or associated with transposons. Cfr-related genes providing similar resistance have been identified in Bacillales, and now also in the pathogens Clostridium difficile and Enterococcus faecium. In addition......, the presence of the cfr gene has been detected in harbours and food markets....

  20. Construction of an efficient biologically contained Pseudomonas putida strain and its survival in outdoor assays

    DEFF Research Database (Denmark)

    Molina, Lazaro; Rodriguez, Cayo Juan Ramos; Ronchel, Maria C.

    1998-01-01

    Active biological containment systems consist of two components, a killing element designed to induce cell death and a control element which modulates the expression of the killing function. We constructed a mini-Tn5 transposon bearing a fusion of the P(lac) promoter to the gef killing gene...

  1. Heat-inducible transgenic expression in the silkmoth Bombyx mori

    Czech Academy of Sciences Publication Activity Database

    Uhlířová, Miroslava; Asahina, Masako; Riddiford, L. M.; Jindra, Marek

    2002-01-01

    Roč. 212, - (2002), s. 145-151 ISSN 0949-944X R&D Projects: GA AV ČR IAB5007002 Institutional research plan: CEZ:AV0Z5007907 Keywords : Germline transformation * piggyBac transposon Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.339, year: 2002

  2. Insertional engineering of chromosomes with Sleeping Beauty transposition: an overview.

    Science.gov (United States)

    Grabundzija, Ivana; Izsvák, Zsuzsanna; Ivics, Zoltán

    2011-01-01

    Novel genetic tools and mutagenesis strategies based on the Sleeping Beauty (SB) transposable element are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of its inherent capacity to insert into DNA, the SB transposon can be developed into powerful tools for chromosomal manipulations. Mutagenesis screens based on SB have numerous advantages including high throughput and easy identification of mutated alleles. Forward genetic approaches based on insertional mutagenesis by engineered SB transposons have the advantage of providing insight into genetic networks and pathways based on phenotype. Indeed, the SB transposon has become a highly instrumental tool to induce tumors in experimental animals in a tissue-specific -manner with the aim of uncovering the genetic basis of diverse cancers. Here, we describe a battery of mutagenic cassettes that can be applied in conjunction with SB transposon vectors to mutagenize genes, and highlight versatile experimental strategies for the generation of engineered chromosomes for loss-of-function as well as gain-of-function mutagenesis for functional gene annotation in vertebrate models.

  3. Proteomics technique opens new frontiers in mobilome research.

    Science.gov (United States)

    Davidson, Andrew D; Matthews, David A; Maringer, Kevin

    2017-01-01

    A large proportion of the genome of most eukaryotic organisms consists of highly repetitive mobile genetic elements. The sum of these elements is called the "mobilome," which in eukaryotes is made up mostly of transposons. Transposable elements contribute to disease, evolution, and normal physiology by mediating genetic rearrangement, and through the "domestication" of transposon proteins for cellular functions. Although 'omics studies of mobilome genomes and transcriptomes are common, technical challenges have hampered high-throughput global proteomics analyses of transposons. In a recent paper, we overcame these technical hurdles using a technique called "proteomics informed by transcriptomics" (PIT), and thus published the first unbiased global mobilome-derived proteome for any organism (using cell lines derived from the mosquito Aedes aegypti ). In this commentary, we describe our methods in more detail, and summarise our major findings. We also use new genome sequencing data to show that, in many cases, the specific genomic element expressing a given protein can be identified using PIT. This proteomic technique therefore represents an important technological advance that will open new avenues of research into the role that proteins derived from transposons and other repetitive and sequence diverse genetic elements, such as endogenous retroviruses, play in health and disease.

  4. PA0305 of Pseudomonas aeruginosa is a quorum quenching acylhomoserine lactone acylase belonging to the Ntn hydrolase superfamily

    NARCIS (Netherlands)

    Wahjudi, Mariana; Papaioannou, Evelina; Hendrawati, Oktavia; van Assen, Aart H. G.; van Merkerk, Ronald; Cool, Robbert H.; Poelarends, Gerrit J.; Quax, Wim

    The Pseudomonas aeruginosa PAO1 genome has at least two genes, pvdQ and quiP, encoding acylhomoserine lactone (AHL) acylases. Two additional genes, pa 1893 and pa0305, have been predicted to encode penicillin acylase proteins, but have not been characterized. Initial studies on a pa0305 transposon

  5. N-Acyl-L-homoserine lactone autoinducers control production of an extracellular lipopeptide biosurfactant required for swarming motility of Serratia liquefaciens MG1

    DEFF Research Database (Denmark)

    Lindum, Peter Wurtz; Anthoni, U; Christophersen, Carsten

    1998-01-01

    A nonswarming Serratia liquefaciens mutant deficient in serrawettin W2 production was constructed by transposon mutagenesis. Sequence homology indicated that insertion had occurred in gene swrA, which encodes a putative peptide synthetase. Expression of swrA is controlled by quorum sensing....

  6. Multiple roles of Pseudomonas aeruginosa TBCF10839 PilY1 in motility, transport and infection

    DEFF Research Database (Denmark)

    Bohn, Yu-Sing Tammy; Brandes, Gudrun; Rakhimova, Elza

    2009-01-01

    Polymorphonuclear neutrophils are the most important mammalian host defence cells against infections with Pseudomonas aeruginosa. Screening of a signature tagged mutagenesis library of the non-piliated P. aeruginosa strain TBCF10839 uncovered that transposon inactivation of its pilY1 gene rendere...

  7. A bacterial genetic screen identifies functional coding sequences of the insect mariner transposable element Famar1 amplified from the genome of the earwig, Forficula auricularia.

    Science.gov (United States)

    Barry, Elizabeth G; Witherspoon, David J; Lampe, David J

    2004-02-01

    Transposons of the mariner family are widespread in animal genomes and have apparently infected them by horizontal transfer. Most species carry only old defective copies of particular mariner transposons that have diverged greatly from their active horizontally transferred ancestor, while a few contain young, very similar, and active copies. We report here the use of a whole-genome screen in bacteria to isolate somewhat diverged Famar1 copies from the European earwig, Forficula auricularia, that encode functional transposases. Functional and nonfunctional coding sequences of Famar1 and nonfunctional copies of Ammar1 from the European honey bee, Apis mellifera, were sequenced to examine their molecular evolution. No selection for sequence conservation was detected in any clade of a tree derived from these sequences, not even on branches leading to functional copies. This agrees with the current model for mariner transposon evolution that expects neutral evolution within particular hosts, with selection for function occurring only upon horizontal transfer to a new host. Our results further suggest that mariners are not finely tuned genetic entities and that a greater amount of sequence diversification than had previously been appreciated can occur in functional copies in a single host lineage. Finally, this method of isolating active copies can be used to isolate other novel active transposons without resorting to reconstruction of ancestral sequences.

  8. Bleomycin resistance : a new dominant selectable marker for plant cell transformation

    NARCIS (Netherlands)

    Hille, Jacques; Verheggen, Frank; Roelvink, Peter; Franssen, Henk; Kammen, Ab van; Zabel, Pim

    1986-01-01

    Plant cells are sensitive to the antibiotic bleomycin, a DNA damaging glycopeptide. A bleomycin resistance determinant, located on transposon Tn5 and functional in bacteria, has been cloned in a plant expression vector and introduced into Nicotiana plumbaginifolia using Agrobacterium tumefaciens.

  9. Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transcription biases and strategies to novel mutant discovery

    Science.gov (United States)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants and it has been implicated as a cause of Crohn’s disease in humans. The generation of comprehensive random mutant banks by transposon mutagenesis is a fundamental wide genomic technology utilized...

  10. [PIWI protein as a nucleolus visitor in Drosophila melanogaster].

    Science.gov (United States)

    Mikhaleva, E A; Iakushev, E Iu; Stoliarenko, A D; Klenov, M S; Pozovskiĭ, Ia M; Gvozdev, V A

    2015-01-01

    The evolutionarily conserved nuclear Piwi protein of Drosophila melanogaster is a representative of the Argonaute small RNA binding protein family. Guided by small piRNAs, Piwi functions in transposon silencing in somatic and germ cells of the gonad. We found that in ovarian somatic and germ cells, as well as in the established ovarian somatic cell line, Piwi is concentrated predominantly in the nucleolus--the main nuclear compartment, participating not only in rRNA synthesis, but also in various cell stress responses. We demonstrated the colocalization of Piwi with nucleolar marker proteins--fibrillarin and Nopp140. A mutation preventing Piwi transport to the nucleus and disturbing transposon silencing (piwi(Nt)) leads to 6-8-fold upregulation of rRNA genes expression, as evaluated by the level of transcripts of transposon insertions in 28S rRNA genes. RNase treatment of live cultured ovarian somatic cells depletes Piwi from the nucleolus. The same effect is observed upon inhibiting RNA polymerase I which transcribes rRNA, but not RNA polymerase II. In contrast, upon heat shock Piwi is concentrated in the nucleolus and is depleted from the nucleoplasm. These results implicate Piwi in RNA polymerase activity modulation and stress response in the nucleolus. We discuss possible noncanonical Piwi functions along with its canonical role in transposon silencing by piRNAs.

  11. An ancient trans-kingdom horizontal transfer of Penelope -like retroelements from arthropods to conifers

    Science.gov (United States)

    Xuan Lin; Nurul Faridi; Claudio Casola

    2016-01-01

    Comparative genomics analyses empowered by the wealth of sequenced genomes have revealed numerous instances of horizontal DNA transfers between distantly related species. In  eukaryotes, repetitive DNA sequences known as transposable elements (TEs) are especially prone to  move across species boundaries. Such horizontal transposon transfers, or HTTs, are relatively  ...

  12. Development of high-throughput phenotyping of metagenomic clones from the human gut microbiome for modulation of eukaryotic cell growth.

    Science.gov (United States)

    Gloux, Karine; Leclerc, Marion; Iliozer, Harout; L'Haridon, René; Manichanh, Chaysavanh; Corthier, Gérard; Nalin, Renaud; Blottière, Hervé M; Doré, Joël

    2007-06-01

    Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.

  13. A comparative phylogenetic analysis of full-length mariner elements

    Indian Academy of Sciences (India)

    Mariner like elements (MLEs) are widely distributed type II transposons with an open reading frame (ORF) for transposase. We studied comparative phylogenetic evolution and inverted terminal repeat (ITR) conservation of MLEs from Indian saturniid silkmoth, Antheraea mylitta with other full length MLEs submitted in the ...

  14. Public Health Risks of Enterobacterial Isolates Producing Extended-Spectrum β-Lactamases or AmpC β-Lactamases in Food and Food-Producing Animals: An EU Perspective of Epidemiology, Analytical Methods, Risk Factors, and Control Options

    DEFF Research Database (Denmark)

    Liebana, Ernesto; Carattoli, Alessandra; Coque, Teresa M.

    2013-01-01

    The blaESBL and blaAmpC genes are spread by plasmid-mediated integrons, insertion sequences, and transposons, some of which are homologous in food animals and humans. Cephalosporin usage in animal production is an important risk factor; restricting such use would be an effective control option....

  15. Pigments influence the tolerance of Pseudomonas aeruginosa PAO1 to photodynamically induced oxidative stress

    DEFF Research Database (Denmark)

    Orlandi, Viviana T; Bolognese, Fabrizio; Chiodaroli, Luca

    2015-01-01

    by exogenous photosensitizers and visible light. To evaluate whether P. aeruginosa pigments can contribute to its relative tolerance to PDT, we analysed the response to this treatment of isogenic transposon mutants of P. aeruginosa PAO1 with altered pigmentation. In general, in the presence of pigments...

  16. IS21-558 insertion sequences are involved in the mobility of the multiresistance gene cfr

    DEFF Research Database (Denmark)

    Kehrenberg, Corinna; Aarestrup, Frank Møller; Schwarz, Stefan

    2007-01-01

    During a study of florfenicol-resistant porcine staphylococci from Denmark, the genes cfr and fexA were detected in the chromosomal DNA or on plasmids of Staphylococcus hyicus, Staphylococcus warneri, and Staphylococcus simulans. A novel variant of the phenicol resistance transposon Tn558...

  17. Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells

    OpenAIRE

    Kebriaei, Partow; Singh, Harjeet; Huls, M. Helen; Figliola, Matthew J.; Bassett, Roland; Olivares, Simon; Jena, Bipulendu; Dawson, Margaret J.; Kumaresan, Pappanaicken R.; Su, Shihuang; Maiti, Sourindra; Dai, Jianliang; Moriarity, Branden; Forget, Marie-Andrée; Senyukov, Vladimir

    2016-01-01

    BACKGROUND. T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR.

  18. Genome-Wide Mutagenesis in Borrelia burgdorferi.

    Science.gov (United States)

    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

  19. Germline transformation of the Mediterranean fruit fly, Ceratitis capitata

    International Nuclear Information System (INIS)

    McCombs, Susan D.

    2000-01-01

    Gene transfer methodology for insects was first developed in Drosophila melanogaster Meigen using a transposon-mediated system based on the P element (Spradling and Rubin 1982, Rubin and Spradling 1982). In addition to the P element, three unrelated transposons have been used successfully in genetic transformation of D. melanogaster: hobo (Blackman et al. 1989), Minos (Loukeris et al. 1992), and mariner (Lidholm et al. 1993). Routine gene transfer in Drosophila created a great deal of optimism amongst researchers who sought to employ transgenic techniques in other arthropods. However, what followed were years of consistently disappointing results in other insect species. For example, the P element system was tried unsuccessfully in several species, but was eventually shown to be non-functional outside the genus Drosophila (O'Brochta and Handler 1988). Ensuing research in non-drosophilids emphasised testing of other Drosophila systems and development of transposons isolated from other species. After nearly 15 years of intensive effort, the first successes have only recently been reported. Three Drosophila-derived transposon-based systems: hobo from D. melanogaster, mariner from Drosophila mauritiana Tsacas and David and Minos from Drosophila hydei Sturtevant have produced germline transformation in Drosophila virilis Sturtevant (Gomez and Handler 1997, Lozovskaya et al. 1996), Aedes aegypti L. (Coates et al. 1998), and Ceratitis capitata (Wied.) (Loukeris et al. 1995), respectively. Germline transformation was accomplished with two transposon-based systems from non-drosophilids, Hermes from Musca domestica L. and piggyBac from Trichoplusia ni Huebner in A. aegypti and C. capitata, respectively

  20. Identification of genetic loci required for Campylobacter resistance to fowlicidin-1, a chicken host defense peptide

    Directory of Open Access Journals (Sweden)

    Ky Van Hoang

    2012-03-01

    Full Text Available Antimicrobial peptides (AMPs are critical components of host defense limiting bacterial infections at the gastrointestinal mucosal surface. Bacterial pathogens have co-evolved with host innate immunity and developed means to counteract the effect of endogenous AMPs. However, molecular mechanisms of AMP resistance in Campylobacter, an important human food borne pathogen with poultry as a major reservoir, are still largely unknown. In this study, random transposon mutagenesis and targeted site-directed mutagenesis approaches were used to identify genetic loci contributing Campylobacter resistance to fowlicidin-1, a chicken AMP belonging to cathelicidin family. An efficient transposon mutagenesis approach (EZ::TNTM Transposome in conjunction with a microtiter plate screening identified three mutants whose susceptibilities to fowlicidin-1 were significantly increased. Backcrossing of the transposon mutations into parent strain confirmed that the AMP-sensitive phenotype in each mutant was linked to the specific transposon insertion. Direct sequencing showed that these mutants have transposon inserted in the genes encoding two-component regulator CbrR, transporter CjaB, and putative trigger factor Tig. Genomic analysis also revealed an operon (Cj1580c-1584c that is homologous to sapABCDF, an operon conferring resistance to AMP in other pathogens. Insertional inactivation of Cj1583c (sapB significantly increased susceptibility of Campylobacter to fowlicidin-1. The sapB as well as tig and cjaB mutants were significantly impaired in their ability to compete with their wild-type strain 81-176 to colonize the chicken cecum. Together, this study identified four genetic loci in Campylobacter that will be useful for characterizing molecular basis of Campylobacter resistance to AMPs, a significant knowledge gap in Campylobacter pathogenesis.

  1. Genome sequence of the endosymbiont Rickettsia peacockii and comparison with virulent Rickettsia rickettsii: identification of virulence factors.

    Directory of Open Access Journals (Sweden)

    Roderick F Felsheim

    2009-12-01

    Full Text Available Rickettsia peacockii, also known as the East Side Agent, is a non-pathogenic obligate intracellular bacterium found as an endosymbiont in Dermacentor andersoni ticks in the western USA and Canada. Its presence in ticks is correlated with reduced prevalence of Rickettsia rickettsii, the agent of Rocky Mountain Spotted Fever. It has been proposed that a virulent SFG rickettsia underwent changes to become the East Side Agent. We determined the genome sequence of R. peacockii and provide a comparison to a closely related virulent R. rickettsii. The presence of 42 chromosomal copies of the ISRpe1 transposon in the genome of R. peacockii is associated with a lack of synteny with the genome of R. rickettsii and numerous deletions via recombination between transposon copies. The plasmid contains a number of genes from distantly related organisms, such as part of the glycosylation island of Pseudomonas aeruginosa. Genes deleted or mutated in R. peacockii which may relate to loss of virulence include those coding for an ankyrin repeat containing protein, DsbA, RickA, protease II, OmpA, ScaI, and a putative phosphoethanolamine transferase. The gene coding for the ankyrin repeat containing protein is especially implicated as it is mutated in R. rickettsii strain Iowa, which has attenuated virulence. Presence of numerous copies of the ISRpe1 transposon, likely acquired by lateral transfer from a Cardinium species, are associated with extensive genomic reorganization and deletions. The deletion and mutation of genes possibly involved in loss of virulence have been identified by this genomic comparison. It also illustrates that the introduction of a transposon into the genome can have varied effects; either correlating with an increase in pathogenicity as in Francisella tularensis or a loss of pathogenicity as in R. peacockii and the recombination enabled by multiple transposon copies can cause significant deletions in some genomes while not in others.

  2. Statistical thermodynamics of aligned rigid rods with attractive lateral interactions: Theory and Monte Carlo simulations

    Science.gov (United States)

    dos Santos, G. J.; Linares, D. H.; Ramirez-Pastor, A. J.

    2018-04-01

    The phase behaviour of aligned rigid rods of length k (k-mers) adsorbed on two-dimensional square lattices has been studied by Monte Carlo (MC) simulations and histogram reweighting technique. The k-mers, containing k identical units (each one occupying a lattice site) were deposited along one of the directions of the lattice. In addition, attractive lateral interactions were considered. The methodology was applied, particularly, to the study of the critical point of the condensation transition occurring in the system. The process was monitored by following the fourth order Binder cumulant as a function of temperature for different lattice sizes. The results, obtained for k ranging from 2 to 7, show that: (i) the transition coverage exhibits a decreasing behaviour when it is plotted as a function of the k-mer size and (ii) the transition temperature, Tc, exhibits a power law dependence on k, Tc ∼k 0 , 4, shifting to higher values as k increases. Comparisons with an analytical model based on a generalization of the Bragg-Williams approximation (BWA) were performed in order to support the simulation technique. A significant qualitative agreement was obtained between BWA and MC results.

  3. Development of DNA affinity techniques for the functional characterization of purified RNA polymerase II transcription factors

    International Nuclear Information System (INIS)

    Garfinkel, S.; Thompson, J.A.; Cohen, R.B.; Brendler, T.; Safer, B.

    1987-01-01

    Affinity adsorption, precipitation, and partitioning techniques have been developed to purify and characterize RNA Pol II transcription components from whole cell extracts (WCE) (HeLa) and nuclear extracts (K562). The titration of these extracts with multicopy constructs of the Ad2 MLP but not pUC8, inhibits transcriptional activity. DNA-binding factors precipitated by this technique are greatly enriched by centrifugation. Using this approach, factors binding to the upstream promoter sequence (UPS) of the Ad2 MLP have been rapidly isolated by Mono Q, Mono S, and DNA affinity chromatography. By U.V. crosslinking to nucleotides containing specific 32 P-phosphodiester bonds within the recognition sequence, this factor is identified as a M/sub r/ = 45,000 polypeptide. To generate an assay system for the functional evaluation of single transcription components, a similar approach using synthetic oligonucleotide sequences spanning single promoter binding sites has been developed. The addition of a synthetic 63-mer containing the UPS element of the Ad2 MLP to HeLa WCE inhibited transcription by 60%. The addition of partially purified UPS binding protein, but not RNA Pol II, restored transcriptional activity. The addition of synthetic oligonucleotides containing other regulatory sequences not present in the Ad2 MLP was without effect

  4. Steps in the development of a Vibrio cholerae El Tor biofilm

    Science.gov (United States)

    Watnick, Paula I.; Kolter, Roberto

    2010-01-01

    Summary We report that, in a simple, static culture system, wild-type Vibrio cholerae El Tor forms a three-dimensional biofilm with characteristic water channels and pillars of bacteria. Furthermore, we have isolated and characterized transposon insertion mutants of V. cholerae that are defective in biofilm development. The transposons were localized to genes involved in (i) the biosynthesis and secretion of the mannose-sensitive haemagglutinin type IV pilus (MSHA); (ii) the synthesis of exopolysaccharide; and (iii) flagellar motility. The phenotypes of these three groups suggest that the type IV pilus and flagellum accelerate attachment to the abiotic surface, the flagellum mediates spread along the abiotic surface, and exopolysaccharide is involved in the formation of three-dimensional biofilm architecture. PMID:10564499

  5. A mutation in the aroE gene affects pigment production, virulence, and chemotaxis in Xanthomonas oryzae pv. oryzae.

    Science.gov (United States)

    Kim, Hong-Il; Noh, Tae-Hwan; Lee, Chang-Soo; Park, Young-Jin

    2015-01-01

    Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB) in rice. To study its function, a random insertion mutation library of Xoo was constructed using the Tn5 transposon. A mutant strain with decreased virulence against the susceptible rice cultivar IR24 was isolated from the library (aroE mutant), which also had extremely low pigment production. Thermal asymmetric interlaced-polymerase chain reaction (TAIL-PCR) and sequence analysis of the mutant revealed that the transposon was inserted into the aroE gene (encoding shikimate dehydrogenase). To investigate gene expression changes in the pigment- and virulence-deficient mutant, DNA microarray analysis was performed, which showed downregulation of 20 genes involved in the chemotaxis of Xoo. Our findings reveal that mutation of the aroE gene affects virulence and pigment production, as well as expression of genes involved in Xoo chemotaxis. Copyright © 2014 Elsevier GmbH. All rights reserved.

  6. Chemical and UV Mutagenesis.

    Science.gov (United States)

    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.

  7. CRISPR/Cas and Cmr modules, mobility and evolution of adaptive immune systems

    DEFF Research Database (Denmark)

    Shah, Shiraz Ali; Garrett, Roger Antony

    2011-01-01

    CRISPR/Cas and CRISPR/Cmr immune machineries of archaea and bacteria provide an adaptive and effective defence mechanism directed specifically against viruses and plasmids. Present data suggest that both CRISPR/Cas and Cmr modules can behave like integral genetic elements. They tend to be located...... in the more variable regions of chromosomes and are displaced by genome shuffling mechanisms including transposition. CRISPR loci may be broken up and dispersed in chromosomes by transposons with the potential for creating genetic novelty. Both CRISPR/Cas and Cmr modules appear to exchange readily between...... the significant barriers imposed by their differing conjugative, transcriptional and translational mechanisms. There are parallels between the CRISPR crRNAs and eukaryal siRNAs, most notably to germ cell piRNAs which are directed, with the help of effector proteins, to silence or destroy transposons...

  8. Invariability of Central Metabolic Flux Distribution in Shewanella oneidensis MR-1 Under Environmental or Genetic Perturbations

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Yinjie; Martin, Hector Garcia; Deutschbauer, Adam; Feng, Xueyang; Huang, Rick; Llora, Xavier; Arkin, Adam; Keasling, Jay D.

    2009-04-21

    An environmentally important bacterium with versatile respiration, Shewanella oneidensis MR-1, displayed significantly different growth rates under three culture conditions: minimal medium (doubling time {approx} 3 hrs), salt stressed minimal medium (doubling time {approx} 6 hrs), and minimal medium with amino acid supplementation (doubling time {approx}1.5 hrs). {sup 13}C-based metabolic flux analysis indicated that fluxes of central metabolic reactions remained relatively constant under the three growth conditions, which is in stark contrast to the reported significant changes in the transcript and metabolite profiles under various growth conditions. Furthermore, ten transposon mutants of S. oneidensis MR-1 were randomly chosen from a transposon library and their flux distributions through central metabolic pathways were revealed to be identical, even though such mutational processes altered the secondary metabolism, for example, glycine and C1 (5,10-Me-THF) metabolism.

  9. Meeting Report: The Role of the Mobilome in Cancer.

    Science.gov (United States)

    Ardeljan, Daniel; Taylor, Martin S; Burns, Kathleen H; Boeke, Jef D; Espey, Michael Graham; Woodhouse, Elisa C; Howcroft, Thomas Kevin

    2016-08-01

    Approximately half of the human genome consists of repetitive sequence attributed to the activities of mobile DNAs, including DNA transposons, RNA transposons, and endogenous retroviruses. Of these, only long interspersed elements (LINE-1 or L1) and sequences copied by LINE-1 remain mobile in our species today. Although cells restrict L1 activity by both transcriptional and posttranscriptional mechanisms, L1 derepression occurs in developmental and pathologic contexts, including many types of cancers. However, we have limited knowledge of the extent and consequences of L1 expression in premalignancies and cancer. Participants in this NIH strategic workshop considered key questions to enhance our understanding of mechanisms and roles the mobilome may play in cancer biology. Cancer Res; 76(15); 4316-9. ©2016 AACR. ©2016 American Association for Cancer Research.

  10. Improved injection needles facilitate germline transformation of the buckeye butterfly Junonia coenia.

    Science.gov (United States)

    Beaudette, Kahlia; Hughes, Tia M; Marcus, Jeffrey M

    2014-01-01

    Germline transformation with transposon vectors is an important tool for insect genetics, but progress in developing transformation protocols for butterflies has been limited by high post-injection ova mortality. Here we present an improved glass injection needle design for injecting butterfly ova that increases survival in three Nymphalid butterfly species. Using the needles to genetically transform the common buckeye butterfly Junonia coenia, the hatch rate for injected Junonia ova was 21.7%, the transformation rate was 3%, and the overall experimental efficiency was 0.327%, a substantial improvement over previous results in other butterfly species. Improved needle design and a higher efficiency of transformation should permit the deployment of transposon-based genetic tools in a broad range of less fecund lepidopteran species.

  11. Transposable Elements in Human Cancer: Causes and Consequences of Deregulation

    Science.gov (United States)

    Anwar, Sumadi Lukman; Wulaningsih, Wahyu; Lehmann, Ulrich

    2017-01-01

    Transposable elements (TEs) comprise nearly half of the human genome and play an essential role in the maintenance of genomic stability, chromosomal architecture, and transcriptional regulation. TEs are repetitive sequences consisting of RNA transposons, DNA transposons, and endogenous retroviruses that can invade the human genome with a substantial contribution in human evolution and genomic diversity. TEs are therefore firmly regulated from early embryonic development and during the entire course of human life by epigenetic mechanisms, in particular DNA methylation and histone modifications. The deregulation of TEs has been reported in some developmental diseases, as well as for different types of human cancers. To date, the role of TEs, the mechanisms underlying TE reactivation, and the interplay with DNA methylation in human cancers remain largely unexplained. We reviewed the loss of epigenetic regulation and subsequent genomic instability, chromosomal aberrations, transcriptional deregulation, oncogenic activation, and aberrations of non-coding RNAs as the potential mechanisms underlying TE deregulation in human cancers. PMID:28471386

  12. Transposable Elements in Human Cancer: Causes and Consequences of Deregulation

    Directory of Open Access Journals (Sweden)

    Sumadi Lukman Anwar

    2017-05-01

    Full Text Available Transposable elements (TEs comprise nearly half of the human genome and play an essential role in the maintenance of genomic stability, chromosomal architecture, and transcriptional regulation. TEs are repetitive sequences consisting of RNA transposons, DNA transposons, and endogenous retroviruses that can invade the human genome with a substantial contribution in human evolution and genomic diversity. TEs are therefore firmly regulated from early embryonic development and during the entire course of human life by epigenetic mechanisms, in particular DNA methylation and histone modifications. The deregulation of TEs has been reported in some developmental diseases, as well as for different types of human cancers. To date, the role of TEs, the mechanisms underlying TE reactivation, and the interplay with DNA methylation in human cancers remain largely unexplained. We reviewed the loss of epigenetic regulation and subsequent genomic instability, chromosomal aberrations, transcriptional deregulation, oncogenic activation, and aberrations of non-coding RNAs as the potential mechanisms underlying TE deregulation in human cancers.

  13. Development of identification process for insect group using radiation marker DNA

    International Nuclear Information System (INIS)

    Muraji, M.; Tamura, T.

    2004-01-01

    Detection of a band pattern for insect groups was tried by using radiation marked DNA clone. A rapid segregation process for poly-type DNA segment was investigated. A band pattern of silkworm was detected by analysis using DNA type transposon, K1.4. The exon regions on genes of hemiptera insect were segregated by in vitro cloning. Band patterns of the silkworm and the other insects were detected by identification process of DNA clone and radiation marker. Family singularity mutation existed in the inserted position of transposon. The family of insect was identified easily by the difference of the detection band patterns. Effective band pattern for family discrimination were obtained by analysis for a part of mitochondria DNA and ribosomal DNA. DNA segregation process was investigated by using the enriched library, also. (M. Suetake)

  14. Genotype-dependent Burst of Transposable Element Expression in Crowns of Hexaploid Wheat (Triticum aestivum L. during Cold Acclimation

    Directory of Open Access Journals (Sweden)

    Debbie Laudencia-Chingcuanco

    2012-01-01

    Full Text Available The expression of 1,613 transposable elements (TEs represented in the Affymetrix Wheat Genome Chip was examined during cold treatment in crowns of four hexaploid wheat genotypes that vary in tolerance to cold and in flowering time. The TE expression profiles showed a constant level of expression throughout the experiment in three of the genotypes. In winter Norstar, the most cold-hardy of the four genotypes, a subset of the TEs showed a burst of expression after vernalization saturation was achieved. About 47% of the TEs were expressed, and both Class I (retrotransposons and Class II (DNA transposons types were well represented. Gypsy and Copia were the most represented among the retrotransposons while CACTA and Mariner were the most represented DNA transposons. The data suggests that the Vrn-A1 region plays a role in the stage-specific induction of TE expression in this genotype.

  15. Simultaneous Identification of Potential Pathogenicity Factors of Mycoplasma agalactiae in the Natural Ovine Host by Negative Selection

    OpenAIRE

    Hegde, Shivanand; Hegde, Shrilakshmi; Zimmermann, Martina; Flöck, Martina; Spergser, Joachim; Rosengarten, Renate; Chopra-Dewasthaly, Rohini

    2015-01-01

    Mycoplasmas possess complex pathogenicity determinants that are largely unknown at the molecular level. Mycoplasma agalactiae serves as a useful model to study the molecular basis of mycoplasma pathogenicity. The generation and in vivo screening of a transposon mutant library of M. agalactiae were employed to unravel its host colonization factors. Tn4001mod mutants were sequenced using a novel sequencing method, and functionally heterogeneous pools containing 15 to 19 selected mutants were sc...

  16. Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

    OpenAIRE

    Karolina Chwialkowska; Urszula Korotko; Joanna Kosinska; Iwona Szarejko; Miroslaw Kwasniewski

    2017-01-01

    Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing ...

  17. Distribution of the multidrug resistance gene cfr in Staphylococcus species isolates from swine farms in China.

    Science.gov (United States)

    Wang, Yang; Zhang, Wanjiang; Wang, Juan; Wu, Congming; Shen, Zhangqi; Fu, Xiao; Yan, Yang; Zhang, Qijing; Schwarz, Stefan; Shen, Jianzhong

    2012-03-01

    A total of 149 porcine Staphylococcus isolates with florfenicol MICs of ≥ 16 μg/ml were screened for the presence of the multiresistance gene cfr, its location on plasmids, and its genetic environment. In total, 125 isolates carried either cfr (16 isolates), fexA (92 isolates), or both genes (17 isolates). The 33 cfr-carrying staphylococci, which included isolates of the species Staphylococcus cohnii, S. arlettae, and S. saprophyticus in which the cfr gene has not been described before, exhibited a wide variety of SmaI pulsed-field gel electrophoresis patterns. In 18 cases, the cfr gene was located on plasmids. Four different types of cfr-carrying plasmids--pSS-01 (n = 2; 40 kb), pSS-02 (n = 3; 35.4 kb), pSS-03 (n = 10; 7.1 kb), and pBS-01 (n = 3; 16.4 kb)--were differentiated on the basis of their sizes, restriction patterns, and additional resistance genes. Sequence analysis revealed that in plasmid pSS-01, the cfr gene was flanked in the upstream part by a complete aacA-aphD-carrying Tn4001-like transposon and in the downstream part by a complete fexA-carrying transposon Tn558. In plasmid pSS-02, an insertion sequence IS21-558 and the cfr gene were integrated into transposon Tn558 and thereby truncated the tnpA and tnpB genes. The smallest cfr-carrying plasmid pSS-03 carried the macrolide-lincosamide-streptogramin B resistance gene erm(C). Plasmid pBS-01, previously described in Bacillus spp., harbored a Tn917-like transposon, including the macrolide-lincosamide-streptogramin B resistance gene erm(B) in the cfr downstream region. Plasmids, which in part carry additional resistance genes, seem to play an important role in the dissemination of the gene cfr among porcine staphylococci.

  18. Complete Genome Sequence of Mycoplasma hominis Strain Sprott (ATCC 33131), Isolated from a Patient with Nongonococcal Urethritis.

    Science.gov (United States)

    Calcutt, Michael J; Foecking, Mark F

    2015-07-09

    Presented here is the complete and annotated genome sequence of Mycoplasma hominis Sprott (ATCC 33131). The chromosome comprises 695,214 bp, which is approximately 30 kb larger than the syntenic genome of M. hominis PG21(T). Tetracycline resistance of strain Sprott is most probably conferred by the tetM determinant, harbored on a mosaic transposon-like structure. Copyright © 2015 Calcutt and Foecking.

  19. Programmed Rearrangement in Ciliates: Paramecium.

    Science.gov (United States)

    Betermier, Mireille; Duharcourt, Sandra

    2014-12-01

    Programmed genome rearrangements in the ciliate Paramecium provide a nice illustration of the impact of transposons on genome evolution and plasticity. During the sexual cycle, development of the somatic macronucleus involves elimination of ∼30% of the germline genome, including repeated DNA (e.g., transposons) and ∼45,000 single-copy internal eliminated sequences (IES). IES excision is a precise cut-and-close process, in which double-stranded DNA cleavage at IES ends depends on PiggyMac, a domesticated piggyBac transposase. Genome-wide analysis has revealed that at least a fraction of IESs originate from Tc/mariner transposons unrelated to piggyBac. Moreover, genomic sequences with no transposon origin, such as gene promoters, can be excised reproducibly as IESs, indicating that genome rearrangements contribute to the control of gene expression. How the system has evolved to allow elimination of DNA sequences with no recognizable conserved motif has been the subject of extensive research during the past two decades. Increasing evidence has accumulated for the participation of noncoding RNAs in epigenetic control of elimination for a subset of IESs, and in trans-generational inheritance of alternative rearrangement patterns. This chapter summarizes our current knowledge of the structure of the germline and somatic genomes for the model species Paramecium tetraurelia, and describes the DNA cleavage and repair factors that constitute the IES excision machinery. We present an overview of the role of specialized RNA interference machineries and their associated noncoding RNAs in the control of DNA elimination. Finally, we discuss how RNA-dependent modification and/or remodeling of chromatin may guide PiggyMac to its cognate cleavage sites.

  20. Metabolic niche of a prominent sulfate-reducing human gut bacterium

    OpenAIRE

    Rey, Federico E.; Gonzalez, Mark D.; Cheng, Jiye; Wu, Meng; Ahern, Philip P.; Gordon, Jeffrey I.

    2013-01-01

    Sulfate-reducing bacteria (SRB) colonize the guts of ∼50% of humans. We used genome-wide transposon mutagenesis and insertion-site sequencing, RNA-Seq, plus mass spectrometry to characterize genetic and environmental factors that impact the niche of Desulfovibrio piger, the most common SRB in a surveyed cohort of healthy US adults. Gnotobiotic mice were colonized with an assemblage of sequenced human gut bacterial species with or without D. piger and fed diets with different levels and types ...

  1. Mobile Genetic Elements and Evolution of CRISPR-Cas Systems: All the Way There and Back

    Science.gov (United States)

    Makarova, Kira S.

    2017-01-01

    Abstract The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) systems of bacterial and archaeal adaptive immunity show multifaceted evolutionary relationships with at least five classes of mobile genetic elements (MGE). First, the adaptation module of CRISPR-Cas that is responsible for the formation of the immune memory apparently evolved from a Casposon, a self-synthesizing transposon that employs the Cas1 protein as the integrase and might have brought additional cas genes to the emerging immunity loci. Second, a large subset of type III CRISPR-Cas systems recruited a reverse transcriptase from a Group II intron, providing for spacer acquisition from RNA. Third, effector nucleases of Class 2 CRISPR-Cas systems that are responsible for the recognition and cleavage of the target DNA were derived from transposon-encoded TnpB nucleases, most likely, on several independent occasions. Fourth, accessory nucleases in some variants of types I and III toxin and type VI effectors RNases appear to be ultimately derived from toxin nucleases of microbial toxin–antitoxin modules. Fifth, the opposite direction of evolution is manifested in the recruitment of CRISPR-Cas systems by a distinct family of Tn7-like transposons that probably exploit the capacity of CRISPR-Cas to recognize unique DNA sites to facilitate transposition as well as by bacteriophages that employ them to cope with host defense. Additionally, individual Cas proteins, such as the Cas4 nuclease, were recruited by bacteriophages and transposons. The two-sided evolutionary connection between CRISPR-Cas and MGE fits the “guns for hire” paradigm whereby homologous enzymatic machineries, in particular nucleases, are shuttled between MGE and defense systems and are used alternately as means of offense or defense. PMID:28985291

  2. Genetic analysis of an Escherichia coli urease locus: evidence of DNA rearrangement.

    OpenAIRE

    Collins, C M; Falkow, S

    1988-01-01

    Ureolytic Escherichia coli strains are uncommon clinical isolates. The urease phenotype in a large percentage of these isolates is unstable and lost upon storage. We examined two urease-positive uropathogenic E. coli isolates that give off urease-negative segregants and determined that the urease phenotype was chromosomally encoded. The urease phenotype was cloned from E. coli 1021 and found to be encoded on a 9.4-kilobase HindIII restriction fragment. Transposon mutagenesis indicated that at...

  3. Widespread dissemination of class 1 integron components in soils and related ecosystems as revealed by cultivation-independent analysis

    OpenAIRE

    Jechalke, Sven; Schreiter, Susanne; Wolters, Birgit; Dealtry, Simone; Heuer, Holger; Smalla, Kornelia

    2014-01-01

    Class 1 integrons contribute to the emerging problem of antibiotic resistance in human medicine by acquisition, exchange, and expression of resistance genes embedded within gene cassettes. Besides the clinical setting they were recently reported from environmental habitats and often located on plasmids and transposons, facilitating their transfer and spread within bacterial communities. In this study we aimed to provide insights into the occurrence of genes typically associated with the class...

  4. The Effectiveness of the Removal of DNA from the Surfaces of EVA-Based Mouthguards using Standard Cleaning Protocols

    Science.gov (United States)

    2016-06-16

    true due to the fact that mouth guards are often used by athletes and military personnel in outdoor settings and given the fact that soil microbes...contact, DNA is transferred from plasmids or transposons. The genetic material transferred is often beneficial to the recipient with possible benefits of...oral injuries. J Am Dent Assoc 2006;137(12):1712-20. 3. Center for Science information, ADA Science Institute, ADA.org/oral health topics/. November

  5. Genome-wide target profiling of piggyBac and Tol2 in HEK 293: pros and cons for gene discovery and gene therapy

    Science.gov (United States)

    2011-01-01

    Background DNA transposons have emerged as indispensible tools for manipulating vertebrate genomes with applications ranging from insertional mutagenesis and transgenesis to gene therapy. To fully explore the potential of two highly active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have conducted a side-by-side comparison of the two transposon systems in the same setting to evaluate their advantages and disadvantages for use in gene therapy and gene discovery. Results We have observed that (1) the Tol2 transposase (but not piggyBac) is highly sensitive to molecular engineering; (2) the piggyBac donor with only the 40 bp 3'-and 67 bp 5'-terminal repeat domain is sufficient for effective transposition; and (3) a small amount of piggyBac transposases results in robust transposition suggesting the piggyBac transpospase is highly active. Performing genome-wide target profiling on data sets obtained by retrieving chromosomal targeting sequences from individual clones, we have identified several piggyBac and Tol2 hotspots and observed that (4) piggyBac and Tol2 display a clear difference in targeting preferences in the human genome. Finally, we have observed that (5) only sites with a particular sequence context can be targeted by either piggyBac or Tol2. Conclusions The non-overlapping targeting preference of piggyBac and Tol2 makes them complementary research tools for manipulating mammalian genomes. PiggyBac is the most promising transposon-based vector system for achieving site-specific targeting of therapeutic genes due to the flexibility of its transposase for being molecularly engineered. Insights from this study will provide a basis for engineering piggyBac transposases to achieve site-specific therapeutic gene targeting. PMID:21447194

  6. Aureusimines in Staphylococcus aureus are not involved in virulence.

    Science.gov (United States)

    Sun, Fei; Cho, Hoonsik; Jeong, Do-Won; Li, Chunling; He, Chuan; Bae, Taeok

    2010-12-29

    Recently, dipeptide aureusimines were reported to activate expression of staphylococcal virulence genes, such as alpha-hemolysin, and increase S. aureus virulence. Surprisingly, most of the virulence genes affected by aureusimines form part of the regulon of the SaeRS two component system (TCS), raising the possibility that SaeRS might be directly or indirectly involved in the aureusimine-dependent signaling process. Using HPLC analyses, we confirmed that a transposon mutant of ausA, the gene encoding the aureusimine dipeptide synthesis enzyme, does not produce dipeptides. However, the transposon mutant showed normal hemolysis activity and alpha-hemolysin/SaeP production. Furthermore, the P1 promoter of the sae operon, one of the targets of the SaeRS TCS, showed normal transcription activity. Moreover, in contrast to the original report, the ausA transposon mutant did not exhibit attenuated virulence in an animal infection model. DNA sequencing revealed that the ausA deletion mutant used in the original study has an 83 nt-duplication in saeS. Hemolysis activity of the original mutant was restored by a plasmid carrying the sae operon. A mutant of the sae operon showed elevated resistance to chloramphenicol and erythromycin, two antibiotics widely used during staphylococcal mutagenesis. At 43°C in the presence of erythromycin and aeration, the conditions typically employed for staphylococcal mutagenesis, an saeR transposon mutant grew much faster than a control mutant and the saeR mutant was highly enriched in a mixed culture experiment. Our results show that the previously reported roles of aureusimines in staphylococcal gene regulation and virulence were due to an unintended mutation in saeS, which was likely selected due to elevated resistance of the mutant to environmental stresses. Thus, there is no evidence indicating that the dipeptide aureusimines play a role in sae-mediated virulence factor production or contribute to staphylococcal virulence.

  7. Metabolite Profiling to Characterize Disease-related Bacteria GLUCONATE EXCRETION BY PSEUDOMONAS AERUGINOSA MUTANTS AND CLINICAL ISOLATES FROM CYSTIC FIBROSIS PATIENTS

    OpenAIRE

    Behrends, V; Bell, TJ; Liebeke, M; Cordes-Blauert, A; Ashraf, SN; Nair, C; Zlosnik, JEA; Williams, HD; Bundy, JG

    2013-01-01

    Metabolic footprinting of supernatants has been proposed as a tool for assigning gene function. We used NMR spectroscopy to measure the exometabolome of 86 single-gene transposon insertion mutant strains (mutants from central carbon metabolism and regulatory mutants) of the opportunistic pathogen Pseudomonas aeruginosa, grown on a medium designed to represent the nutritional content of cystic fibrosis sputum. Functionally related genes had similar metabolic profiles. E.g. for two-component sy...

  8. Analysis of the LIV System of Campylobacter jejuni Reveals Alternative Roles for LivJ and LivK in Commensalism beyond Branched-Chain Amino Acid Transport ▿

    OpenAIRE

    Ribardo, Deborah A.; Hendrixson, David R.

    2011-01-01

    Campylobacter jejuni is a leading cause of diarrheal disease in humans and an intestinal commensal in poultry and other agriculturally important animals. These zoonotic infections result in significant amounts of C. jejuni present in the food supply to contribute to disease in humans. We previously found that a transposon insertion in Cjj81176_1038, encoding a homolog of the Escherichia coli LivJ periplasmic binding protein of the leucine, isoleucine, and valine (LIV) branched-chain amino aci...

  9. Production of the Bioactive Compounds Violacein and Indolmycin Is Conditional in a maeA Mutant of Pseudoalteromonas luteoviolacea S4054 Lacking the Malic Enzyme

    DEFF Research Database (Denmark)

    Schmidt Thøgersen, Mariane; Delpin, Marina; Melchiorsen, Jette

    2016-01-01

    cluster was not interrupted by the transposon; instead the insertion was located to the maeA gene encoding the malic enzyme. Supernatant of the mutant strain inhibited Vibrio anguillarum and Staphylococcus aureus in well diffusion assays and in MIC assays at the same level as the wild type strain...... of violacein and indolmycin may be metabolically linked and that yet unidentified antibacterial compound(s) may be play a role in the antibacterial activity of P. luteoviolacea....

  10. What tangled web: barriers to rampant horizontal gene transfer

    OpenAIRE

    Kurland, Charles

    2005-01-01

    Dawkins in his The Selfish Gene(1) quite aptly applies the term "selfish" to parasitic repetitive DNA sequences endemic to eukaryotic genomes, especially vertebrates. Doolittle and Sapienza (2) as well as Orgel and Crick (3) enlivened this notion of selfish DNA with the identification of such repetitive sequences as remnants of mobile elements such as transposons. In addition, Orgel and Crick (3) associated parasitic DNA with a potential to outgrow their host genomes by propagating ...

  11. [Annotation of the mobilomes of nine teleost species].

    Science.gov (United States)

    Gao, Bo; Shen, Dan; Chen, Cai; Wang, Saisai; Yang, Kunlun; Chen, Wei; Wang, Wei; Zhang, Li; Song, Chengyi

    2018-01-25

    In this study, the mobilomes of nine teleost species were annotated by bioinformatics methods. Both of the mobilome size and constitute displayed a significant difference in 9 species of teleost fishes. The species of mobilome content ranking from high to low were zebrafish, medaka, tilapia, coelacanth, platyfish, cod, stickleback, tetradon and fugu. Mobilome content and genome size were positively correlated. The DNA transposons displayed higher diversity and larger variation in teleost (0.50% to 38.37%), was a major determinant of differences in teleost mobilomes, and hAT and Tc/Mariner superfamily were the major DNA transposons in teleost. RNA transposons also exhibited high diversity in teleost, LINE transposons accounted for 0.53% to 5.75% teleost genomic sequences, and 14 superfamilies were detected. L1, L2, RTE and Rex retrotransposons obtained significant amplification. While LTR displayed low amplification in most teleost with less than 2% of genome coverages, except in zebrafish and stickleback, where LTR reachs 5.58% and 2.51% of genome coverages respectively. And 6 LTR superfamilies (Copia, DIRS, ERV, Gypsy, Ngaro and Pao) were detected in the teleost, and Gypsy exhibits obvious amplication among them. While the SINE represents the weakest ampification types in teleost, only within zebrafish and coelacanth, it represents 3.28% and 5.64% of genome coverages, in the other 7 teleost, it occupies less than 1% of genomes, and tRNA, 5S and MIR families of SINE have a certain degree of amplification in some teleosts. This study shows that the teleost display high diversity and large variation of mobilome, there is a strong correlation with the size variations of genomes and mobilome contents in teleost, mobilome is an important factor in determining the teleost genome size.

  12. Insight into the mobilome of Aeromonas strains

    OpenAIRE

    Piotrowska, Marta; Popowska, Magdalena

    2015-01-01

    The mobilome is a pool of genes located within mobile genetic elements (MGE), such as plasmids, IS elements, transposons, genomic/pathogenicity islands, and integron-associated gene cassettes. These genes are often referred to as ?flexible? and may encode virulence factors, toxic compounds as well as resistance to antibiotics. The phenomenon of MGE transfer between bacteria, known as horizontal gene transfer (HGT), is well documented. The genes present on MGE are subject to continuous process...

  13. An abyssal mobilome: Viruses, plasmids and vesicles from deep-sea hydrothermal vents

    OpenAIRE

    Lossouarn, Julien; Dupont, Samuel; Gorlas, Aurore; Mercier, Coraline; Bienvenu, Nadege; Marguet, Evelyne; Forterre, Patrick; Geslin, Claire

    2015-01-01

    Mobile genetic elements (MGEs) such as viruses, plasmids, vesicles, gene transfer agents (GTAs), transposons and transpovirions, which collectively represent the mobilome, interact with cellular organisms from all three domains of life, including those thriving in the most extreme environments. While efforts have been made to better understand deep-sea vent microbial ecology, our knowledge of the mobilome associated with prokaryotes inhabiting deep-sea hydrothermal vents remains limited. Here...

  14. Transposition of Tn5096 from a temperature-sensitive transducible plasmid in Streptomyces spp.

    OpenAIRE

    McHenney, M A; Baltz, R H

    1991-01-01

    Transposon Tn5096 was inserted into a derivative of the temperature-sensitive plasmid pMT660 containing the bacteriophage FP43 pac site. The resulting plasmid, pRHB126, was transduced by FP43 into several Streptomyces species. Tn5096 transposed from pRHB126 into different sites in the genomes of Streptomyces ambofaciens, Streptomyces cinnamonensis, Streptomyces coelicolor A3(2), Streptomyces fradiae, Streptomyces griseofuscus, and Streptomyces thermotolerans.

  15. A Two-Component Regulatory System, CsrR-CsrS, Represses Expression of Three Streptococcus pyogenes Virulence Factors, Hyaluronic Acid Capsule, Streptolysin S, and Pyrogenic Exotoxin B

    OpenAIRE

    Heath, Andrew; DiRita, Victor J.; Barg, Neil L.; Engleberg, N. Cary

    1999-01-01

    Certain Tn916 insertions in the chromosome of an M1-type, nonmucoid Streptococcus pyogenes isolate (MGAS166) were previously shown to result in stable mucoidy with increased expression of the capsular synthetic genes. The transposon insertions in these strains are directly upstream of an apparent operon encoding a two-component regulatory system, designated csrR-csrS. Compared with MGAS166, these mucoid mutants are more hemolytic and cause significantly more tissue damage in a murine model of...

  16. Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance

    DEFF Research Database (Denmark)

    Petersen, M.; Brodersen, P.; Naested, H.

    2000-01-01

    Transposon inactivation of Arabidopsis MAP kinase 4 produced the mpk4 mutant exhibiting constitutive systemic acquired resistance (SAR) including elevated salicylic acid (SA) revels, increased resistance to virulent pathogens, and constitutive pathogenesis-related gene expression shown by Northern...... of NPR1. PDF1.2 and THI2.1 gene induction by jasmonate was blocked in mpk4 expressing NahG, suggesting that MPK4 is required for jasmonic acid-responsive gene expression....

  17. Naringenin Regulates Expression of Genes Involved in Cell Wall Synthesis in Herbaspirillum seropedicae▿

    Science.gov (United States)

    Tadra-Sfeir, M. Z.; Souza, E. M.; Faoro, H.; Müller-Santos, M.; Baura, V. A.; Tuleski, T. R.; Rigo, L. U.; Yates, M. G.; Wassem, R.; Pedrosa, F. O.; Monteiro, R. A.

    2011-01-01

    Five thousand mutants of Herbaspirillum seropedicae SmR1 carrying random insertions of transposon pTnMod-OGmKmlacZ were screened for differential expression of LacZ in the presence of naringenin. Among the 16 mutants whose expression was regulated by naringenin were genes predicted to be involved in the synthesis of exopolysaccharides, lipopolysaccharides, and auxin. These loci are probably involved in establishing interactions with host plants. PMID:21257805

  18. Naringenin regulates expression of genes involved in cell wall synthesis in Herbaspirillum seropedicae.

    Science.gov (United States)

    Tadra-Sfeir, M Z; Souza, E M; Faoro, H; Müller-Santos, M; Baura, V A; Tuleski, T R; Rigo, L U; Yates, M G; Wassem, R; Pedrosa, F O; Monteiro, R A

    2011-03-01

    Five thousand mutants of Herbaspirillum seropedicae SmR1 carrying random insertions of transposon pTnMod-OGmKmlacZ were screened for differential expression of LacZ in the presence of naringenin. Among the 16 mutants whose expression was regulated by naringenin were genes predicted to be involved in the synthesis of exopolysaccharides, lipopolysaccharides, and auxin. These loci are probably involved in establishing interactions with host plants.

  19. Chromosomally Encoded mcr-5 in Colistin non-susceptible Pseudomonas aeruginosa.

    Science.gov (United States)

    Snesrud, Erik; Maybank, Rosslyn; Kwak, Yoon I; Jones, Anthony R; Hinkle, Mary K; Mc Gann, Patrick

    2018-05-29

    Whole genome sequencing (WGS) of historical Pseudomonas aeruginosa clinical isolates identified a chromosomal copy of mcr-5 within a Tn 3 -like transposon in P. aeruginosa MRSN 12280. The isolate was non-susceptible to colistin by broth microdilution and genome analysis revealed no mutations known to confer colistin resistance. To the best of our knowledge, this is the first report of mcr in colistin non-susceptible P. aeruginosa .

  20. Genome Sequences of Subcluster K5 Mycobacteriophages AlleyCat, Edugator, and Guillsminger.

    Science.gov (United States)

    King, Rodney A; Slowan-Pomeroy, Tina M; Thomas, Jodi E; Ahmed, Tithe; Alexander, Katie L; Biddle, James M; Daniels, Makenzie K; Rowlett, Jenna R; Senay, Taylor E; Rinehart, Claire A; Staples, Amanda K; Rowland, Naomi S; Gaffney, Bobby L; Emmons, Christine B; Hauk, Maya D; Nguyen, Rebecca L; Naegele, Leonard; Strickland, Summer S; Briggs, Laura A; Rush, Alexander N; Saha, Sanghamitra; Sadana, Rachna; Cresawn, Steven G; Russell, Daniel A; Garlena, Rebecca A; Pope, Welkin H; Jacobs-Sera, Deborah; Hatfull, Graham F

    2017-11-09

    Bacteriophages AlleyCat, Edugator, and Guillsminger were isolated on Mycobacterium smegmatis mc 2 155 from enriched soil samples. All are members of mycobacteriophage subcluster K5, with genomes of 62,112 to 63,344 bp. Each genome contains 92 to 99 predicted protein-coding genes and one tRNA. Guillsminger is the first mycobacteriophage to carry an IS 1380 family transposon. Copyright © 2017 King et al.

  1. Identification of the Genes Involved in the Biofilm-like Structures on Actinomyces oris K20, a Clinical Isolate from an Apical Lesion

    Science.gov (United States)

    2013-01-01

    bacteria in clinically asymptomatic periapical pathosis. J Endod 1990;16:534–8. 5. Nair PNR. On the causes of persistent apical periodontitis : a review...Identification of the Genes Involved in the Biofilm-like Structures on Actinomyces oris K20, a Clinical Isolate from an Apical Lesion Chiho Mashimo...Actinomyces oris K20. (J Endod 2013;39:44–48) Key Words Actinomyces oris, apical abscess, biofilm, polysac- charide deacetylase, transposon mutagenesis

  2. Aureusimines in Staphylococcus aureus are not involved in virulence.

    Directory of Open Access Journals (Sweden)

    Fei Sun

    2010-12-01

    Full Text Available Recently, dipeptide aureusimines were reported to activate expression of staphylococcal virulence genes, such as alpha-hemolysin, and increase S. aureus virulence. Surprisingly, most of the virulence genes affected by aureusimines form part of the regulon of the SaeRS two component system (TCS, raising the possibility that SaeRS might be directly or indirectly involved in the aureusimine-dependent signaling process.Using HPLC analyses, we confirmed that a transposon mutant of ausA, the gene encoding the aureusimine dipeptide synthesis enzyme, does not produce dipeptides. However, the transposon mutant showed normal hemolysis activity and alpha-hemolysin/SaeP production. Furthermore, the P1 promoter of the sae operon, one of the targets of the SaeRS TCS, showed normal transcription activity. Moreover, in contrast to the original report, the ausA transposon mutant did not exhibit attenuated virulence in an animal infection model. DNA sequencing revealed that the ausA deletion mutant used in the original study has an 83 nt-duplication in saeS. Hemolysis activity of the original mutant was restored by a plasmid carrying the sae operon. A mutant of the sae operon showed elevated resistance to chloramphenicol and erythromycin, two antibiotics widely used during staphylococcal mutagenesis. At 43°C in the presence of erythromycin and aeration, the conditions typically employed for staphylococcal mutagenesis, an saeR transposon mutant grew much faster than a control mutant and the saeR mutant was highly enriched in a mixed culture experiment.Our results show that the previously reported roles of aureusimines in staphylococcal gene regulation and virulence were due to an unintended mutation in saeS, which was likely selected due to elevated resistance of the mutant to environmental stresses. Thus, there is no evidence indicating that the dipeptide aureusimines play a role in sae-mediated virulence factor production or contribute to staphylococcal

  3. Analysis of an ordered, comprehensive STM mutant library in infectious Borrelia burgdorferi: insights into the genes required for mouse infectivity.

    Directory of Open Access Journals (Sweden)

    Tao Lin

    Full Text Available The identification of genes important in the pathogenesis of Lyme disease Borrelia has been hampered by exceedingly low transformation rates in low-passage, infectious organisms. Using the infectious, moderately transformable B. burgdorferi derivative 5A18NP1 and signature-tagged versions of the Himar1 transposon vector pGKT, we have constructed a defined transposon library for the efficient genome-wide investigation of genes required for wild-type pathogenesis, in vitro growth, physiology, morphology, and plasmid replication. To facilitate analysis, the insertion sites of 4,479 transposon mutants were determined by sequencing. The transposon insertions were widely distributed across the entire B. burgdorferi genome, with an average of 2.68 unique insertion sites per kb DNA. The 10 linear plasmids and 9 circular plasmids had insertions in 33 to 100 percent of their predicted genes. In contrast, only 35% of genes in the 910 kb linear chromosome had incapacitating insertions; therefore, the remaining 601 chromosomal genes may represent essential gene candidates. In initial signature-tagged mutagenesis (STM analyses, 434 mutants were examined at multiple tissue sites for infectivity in mice using a semi-quantitative, Luminex-based DNA detection method. Examples of genes found to be important in mouse infectivity included those involved in motility, chemotaxis, the phosphoenolpyruvate phosphotransferase system, and other transporters, as well as putative plasmid maintenance genes. Availability of this ordered STM library and a high-throughput screening method is expected to lead to efficient assessment of the roles of B. burgdorferi genes in the infectious cycle and pathogenesis of Lyme disease.

  4. Advances in DNA methylation: 5-hydroxymethylcytosine revisited

    DEFF Research Database (Denmark)

    Dahl, Christina; Grønbæk, Kirsten; Guldberg, Per

    2011-01-01

    modification involved in gene regulation, X-chromosome inactivation, genomic imprinting, long-term silencing of transposons and cancer development is well described. 5hmC, on the other hand, has only recently entered center stage when it was shown that the Ten-Eleven-Translocation (TET) family of oxygenases...... in cancer development, and developing sequencing methodologies that can accurately distinguish among cytosine, 5mC and 5hmC at single-base-pair resolution....

  5. Proteus genomic island 1 (PGI1), a new resistance genomic island from two Proteus mirabilis French clinical isolates.

    Science.gov (United States)

    Siebor, Eliane; Neuwirth, Catherine

    2014-12-01

    To analyse the genetic environment of the antibiotic resistance genes in two clinical Proteus mirabilis isolates resistant to multiple antibiotics. PCR, gene walking and whole-genome sequencing were used to determine the sequence of the resistance regions, the surrounding genetic structure and the flanking chromosomal regions. A genomic island of 81.1 kb named Proteus genomic island 1 (PGI1) located at the 3'-end of trmE (formerly known as thdF) was characterized. The large MDR region of PGI1 (55.4 kb) included a class 1 integron (aadB and aadA2) and regions deriving from several transposons: Tn2 (blaTEM-135), Tn21, Tn6020-like transposon (aphA1b), a hybrid Tn502/Tn5053 transposon, Tn501, a hybrid Tn1696/Tn1721 transposon [tetA(A)] carrying a class 1 integron (aadA1) and Tn5393 (strA and strB). Several ISs were also present (IS4321, IS1R and IS26). The PGI1 backbone (25.7 kb) was identical to that identified in Salmonella Heidelberg SL476 and shared some identity with the Salmonella genomic island 1 (SGI1) backbone. An IS26-mediated recombination event caused the division of the MDR region into two parts separated by a large chromosomal DNA fragment of 197 kb, the right end of PGI1 and this chromosomal sequence being in inverse orientation. PGI1 is a new resistance genomic island from P. mirabilis belonging to the same island family as SGI1. The role of PGI1 in the spread of antimicrobial resistance genes among Enterobacteriaceae of medical importance needs to be evaluated. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. Genome-Wide Analysis of Salicylate and Dibenzofuran Metabolism in Sphingomonas wittichii RW1

    Directory of Open Access Journals (Sweden)

    Edith eCoronado

    2012-08-01

    Full Text Available Sphingomonas wittichii RW1 is a bacterium isolated for its ability to degrade the xenobiotic compounds dibenzodioxin and dibenzofuran (DBF. A number of genes involved in DBF degradation have been previously characterized, such as the dxn cluster, dbfB, and the electron transfer components fdx1, fdx3 and redA2. Here we use a combination of whole genome transcriptome analysis and transposon library screening to characterize RW1 catabolic and other genes implicated in the reaction to or degradation of DBF. To detect differentially expressed genes upon exposure to DBF, we applied three different growth exposure experiments, using either short DBF exposures to actively growing cells or growing them with DBF as sole carbon and energy source. Genome-wide gene expression was examined using a custom-made microarray. In addition, proportional abundance determination of transposon insertions in RW1 libraries grown on salicylate or DBF by ultra-high throughput sequencing was used to infer genes whose interruption caused a fitness loss for growth on DBF. Expression patterns showed that batch and chemostat growth conditions, and short or long exposure of cells to DBF produced very different responses. Numerous other uncharacterized catabolic gene clusters putatively involved in aromatic compound metabolism increased expression in response to DBF. In addition, only very few transposon insertions completely abolished growth on DBF. Some of those (e.g., in dxnA1 were expected, whereas others (in a gene cluster for phenylacetate degradation were not. Both transcriptomic data and transposon screening suggest operation of multiple redundant and parallel aromatic pathways, depending on DBF exposure. In addition, increased expression of other non-catabolic genes suggests that during initial exposure, S. wittichii RW1 perceives DBF as a stressor, whereas after longer exposure, the compound is recognized as a carbon source and metabolized using several pathways in

  7. Female site-specific transposase-induced recombination: a high-efficiency method for fine mapping mutations on the X chromosome in Drosophila.

    OpenAIRE

    Marcus, Jeffrey M

    2003-01-01

    P-element transposons in the Drosophila germline mobilize only in the presence of the appropriate transposase enzyme. Sometimes, instead of mobilizing completely, P elements will undergo site-specific recombination with the homologous chromosome. Site-specific recombination is the basis for male recombination mapping, since the male germline does not normally undergo recombination. Site-specific recombination also takes place in females, but this has been difficult to study because of the obs...

  8. Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

    Science.gov (United States)

    Metts, J; West, J; Doares, S H; Matthysse, A G

    1991-02-01

    Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was determined by cloning the DNA containing the transposon insertion and using the cloned DNA to replace the wild-type DNA in the parent bacterial strain by marker exchange. The transposon insertions in the three mutants mapped at three widely separated locations on the bacterial chromosome. The effects of the mutations on various steps in tumor formation were examined. All three mutants showed no alteration in binding to carrot cells. However, none of the mutants showed any induction of vir genes by acetosyringone under conditions in which the parent strain showed vir gene induction. When the mutant bacteria were examined for changes in surface components, it was found that all three of the mutants showed a similar alteration in lipopolysaccharide (LPS). LPS from the mutants was larger in size and more heavily saccharide substituted than LPS from the parent strain. Two of the mutants showed no detectable alteration in outer membrane and periplasmic space proteins. The third mutant, Ivr-225, was missing a 79-kDa surface peptide. The reason(s) for the failure of vir gene induction in these mutants and its relationship, if any, to the observed alteration in LPS are unknown.

  9. Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection.

    Science.gov (United States)

    Trubitsyna, Maryia; Michlewski, Gracjan; Finnegan, David J; Elfick, Alistair; Rosser, Susan J; Richardson, Julia M; French, Christopher E

    2017-06-02

    Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Identification of Genes Involved in Polysaccharide-Independent Staphylococcus aureus Biofilm Formation

    Science.gov (United States)

    Boles, Blaise R.; Thoendel, Matthew; Roth, Aleeza J.; Horswill, Alexander R.

    2010-01-01

    Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor α2-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development. PMID:20418950

  11. Identification of genes involved in polysaccharide-independent Staphylococcus aureus biofilm formation.

    Directory of Open Access Journals (Sweden)

    Blaise R Boles

    2010-04-01

    Full Text Available Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor alpha(2-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development.

  12. Minotaur is critical for primary piRNA biogenesis.

    Science.gov (United States)

    Vagin, Vasily V; Yu, Yang; Jankowska, Anna; Luo, Yicheng; Wasik, Kaja A; Malone, Colin D; Harrison, Emily; Rosebrock, Adam; Wakimoto, Barbara T; Fagegaltier, Delphine; Muerdter, Felix; Hannon, Gregory J

    2013-08-01

    Piwi proteins and their associated small RNAs are essential for fertility in animals. In part, this is due to their roles in guarding germ cell genomes against the activity of mobile genetic elements. piRNA populations direct Piwi proteins to silence transposon targets and, as such, form a molecular code that discriminates transposons from endogenous genes. Information ultimately carried by piRNAs is encoded within genomic loci, termed piRNA clusters. These give rise to long, single-stranded, primary transcripts that are processed into piRNAs. Despite the biological importance of this pathway, neither the characteristics that define a locus as a source of piRNAs nor the mechanisms that catalyze primary piRNA biogenesis are well understood. We searched an EMS-mutant collection annotated for fertility phenotypes for genes involved in the piRNA pathway. Twenty-seven homozygous sterile strains showed transposon-silencing defects. One of these, which strongly impacted primary piRNA biogenesis, harbored a causal mutation in CG5508, a member of the Drosophila glycerol-3-phosphate O-acetyltransferase (GPAT) family. These enzymes catalyze the first acylation step on the path to the production of phosphatidic acid (PA). Though this pointed strongly to a function for phospholipid signaling in the piRNA pathway, a mutant form of CG5508, which lacks the GPAT active site, still functions in piRNA biogenesis. We have named this new biogenesis factor Minotaur.

  13. No-Go Zones for Mariner Transposition

    Directory of Open Access Journals (Sweden)

    Brian J. Akerley

    2016-10-01

    Full Text Available The property of transposons to randomly insert into target DNA has long been exploited for generalized mutagenesis and forward genetic screens. Newer applications that monitor the relative abundance of each transposon insertion in large libraries of mutants can be used to evaluate the roles in cellular fitness of all genes of an organism, provided that transposition is in fact random across all genes. In a recent article, Kimura and colleagues identified an important exception to the latter assumption [S. Kimura, T. P. Hubbard, B. M. Davis, M. K. Waldor, mBio 7(4:e01351-16, 2016, doi:10.1128/mBio.01351-16]. They provide evidence that the Mariner transposon exhibits locus-specific site preferences in the presence of the histone-like nucleoid structuring protein H-NS. This effect was shown to bias results for important virulence loci in Vibrio cholerae and to result in misidentification of genes involved in growth in vitro. Fortunately, the bulk of this bacterium’s genome was unaffected by this bias, and recognizing the H-NS effect allows filtering to improve the accuracy of the results.

  14. Candidate Essential Genes in Burkholderia cenocepacia J2315 Identified by Genome-Wide TraDIS

    KAUST Repository

    Wong, Yee-Chin

    2016-08-22

    Burkholderia cenocepacia infection often leads to fatal cepacia syndrome in cystic fibrosis patients. However, antibiotic therapy rarely results in complete eradication of the pathogen due to its intrinsic resistance to many clinically available antibiotics. Recent attention has turned to the identification of essential genes as the proteins encoded by these genes may serve as potential targets for development of novel antimicrobials. In this study, we utilized TraDIS (Transposon Directed Insertion-site Sequencing) as a genome-wide screening tool to facilitate the identification of B. cenocepacia genes essential for its growth and viability. A transposon mutant pool consisting of approximately 500,000 mutants was successfully constructed, with more than 400,000 unique transposon insertion sites identified by computational analysis of TraDIS datasets. The saturated library allowed for the identification of 383 genes that were predicted to be essential in B. cenocepacia. We extended the application of TraDIS to identify conditionally essential genes required for in vitro growth and revealed an additional repertoire of 439 genes to be crucial for B. cenocepacia growth under nutrient-depleted conditions. The library of B. cenocepacia mutants can subsequently be subjected to various biologically related conditions to facilitate the discovery of genes involved in niche adaptation as well as pathogenicity and virulence.

  15. A novel intergenic ETnII-β insertion mutation causes multiple malformations in polypodia mice.

    Directory of Open Access Journals (Sweden)

    Jessica A Lehoczky

    Full Text Available Mouse early transposon insertions are responsible for ~10% of spontaneous mutant phenotypes. We previously reported the phenotypes and genetic mapping of Polypodia, (Ppd, a spontaneous, X-linked dominant mutation with profound effects on body plan morphogenesis. Our new data shows that mutant mice are not born in expected Mendelian ratios secondary to loss after E9.5. In addition, we refined the Ppd genetic interval and discovered a novel ETnII-β early transposon insertion between the genes for Dusp9 and Pnck. The ETn inserted 1.6 kb downstream and antisense to Dusp9 and does not disrupt polyadenylation or splicing of either gene. Knock-in mice engineered to carry the ETn display Ppd characteristic ectopic caudal limb phenotypes, showing that the ETn insertion is the Ppd molecular lesion. Early transposons are actively expressed in the early blastocyst. To explore the consequences of the ETn on the genomic landscape at an early stage of development, we compared interval gene expression between wild-type and mutant ES cells. Mutant ES cell expression analysis revealed marked upregulation of Dusp9 mRNA and protein expression. Evaluation of the 5' LTR CpG methylation state in adult mice revealed no correlation with the occurrence or severity of Ppd phenotypes at birth. Thus, the broad range of phenotypes observed in this mutant is secondary to a novel intergenic ETn insertion whose effects include dysregulation of nearby interval gene expression at early stages of development.

  16. Enhancement of the potential to utilize octopine in the nonfluorescent Pseudomonas sp. strain 92

    International Nuclear Information System (INIS)

    Gill, S.S.; Boivin, R.; Dion, P.

    1991-01-01

    The nonfluorescent Pseudomonas sp. strain 92 requires the presence of a supplementary carbon source for growth on octopine, whereas the spontaneous mutant RB100 has acquired the capacity to utilize this opine as the sole carbon and nitrogen source. Insertional mutagenesis of RB100 with transposon Tn5 generated mutants which were unable to grow on octopine and others which grew slowly on this substrate. Both types of mutants yielded revertants that had regained the ability to utilize octopine. Some of the revertants had lost the transposon, whereas in others the transposon was retained but with rearrangements of the insertion site. Genes of octopine catabolism from strain 92 were cloned on a cosmid vector to generate pK3. The clone pK3 conferred the ability to utilize octopine as the sole carbon and nitrogen source on the host Pseudomonas putida KT2440. Although they conferred an equivalent growth phenotype, the mutant genes carried by RB100 and the cloned genes on pK3 differed in their regulation. Utilization of [ 14 C]octopine was inducible by octopine in RB100 and was constitutive in KT2440(pK3)

  17. Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle

    Directory of Open Access Journals (Sweden)

    Helena Escobar

    2016-01-01

    Full Text Available Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6. Cg-Dysfprmd Prkdcscid/J (Scid/BLA/J mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy.

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

    Science.gov (United States)

    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.

  19. Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis

    Science.gov (United States)

    Chakraborty, Syandan; Ji, HaYeun; Chen, Jack; Gersbach, Charles A.; Leong, Kam W.

    2014-01-01

    Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed “cut-and-paste” mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In this study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the separation of the transposase element from the polyA sequence after transposition leads to its deactivation. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells. PMID:25492703

  20. Two Sets of Piwi Proteins Are Involved in Distinct sRNA Pathways Leading to Elimination of Germline-Specific DNA

    Directory of Open Access Journals (Sweden)

    Dominique I. Furrer

    2017-07-01

    Full Text Available Piwi proteins and piRNAs protect eukaryotic germlines against the spread of transposons. During development in the ciliate Paramecium, two Piwi-dependent sRNA classes are involved in the elimination of transposons and transposon-derived DNA: scan RNAs (scnRNAs, associated with Ptiwi01 and Ptiwi09, and iesRNAs, whose binding partners we now identify as Ptiwi10 and Ptiwi11. scnRNAs derive from the maternal genome and initiate DNA elimination during development, whereas iesRNAs continue DNA targeting until the removal process is complete. Here, we show that scnRNAs and iesRNAs are processed by distinct Dicer-like proteins and bind Piwi proteins in a mutually exclusive manner, suggesting separate biogenesis pathways. We also demonstrate that the PTIWI10 gene is transcribed from the developing nucleus and that its transcription depends on prior DNA excision, suggesting a mechanism of gene expression control triggered by the removal of short DNA segments interrupting the gene.

  1. Distinct functions for the Drosophila piRNA pathway in genome maintenance and telomere protection.

    Directory of Open Access Journals (Sweden)

    Jaspreet S Khurana

    2010-12-01

    Full Text Available Transposons and other selfish DNA elements can be found in all phyla, and mobilization of these elements can compromise genome integrity. The piRNA (PIWI-interacting RNA pathway silences transposons in the germline, but it is unclear if this pathway has additional functions during development. Here we show that mutations in the Drosophila piRNA pathway genes, armi, aub, ago3, and rhi, lead to extensive fragmentation of the zygotic genome during the cleavage stage of embryonic divisions. Additionally, aub and armi show defects in telomere resolution during meiosis and the cleavage divisions; and mutations in lig-IV, which disrupt non-homologous end joining, suppress these fusions. By contrast, lig-IV mutations enhance chromosome fragmentation. Chromatin immunoprecipitation studies show that aub and armi mutations disrupt telomere binding of HOAP, which is a component of the telomere protection complex, and reduce expression of a subpopulation of 19- to 22-nt telomere-specific piRNAs. Mutations in rhi and ago3, by contrast, do not block HOAP binding or production of these piRNAs. These findings uncover genetically separable functions for the Drosophila piRNA pathway. The aub, armi, rhi, and ago3 genes silence transposons and maintain chromosome integrity during cleavage-stage embryonic divisions. However, the aub and armi genes have an additional function in assembly of the telomere protection complex.

  2. Identification of Xylella fastidiosa antivirulence genes: hemagglutinin adhesins contribute a biofilm maturation to X. fastidios and colonization and attenuate virulence.

    Science.gov (United States)

    Guilhabert, Magalie R; Kirkpatrick, Bruce C

    2005-08-01

    Xylella fastidosa, a gram-negative, xylem-limited bacterium, is the causal agent of several economically important plant diseases, including Pierce's disease (PD) and citrus variegated chlorosis (CVC). Until recently, the inability to transform or produce transposon mutants of X. fastidosa had been a major impediment to identifying X. fastidosa genes that mediate pathogen and plant interactions. A random transposon (Tn5) library of X. fastidosa was constructed and screened for mutants showing more severe symptoms and earlier grapevine death (hypervirulence) than did vines infected with the wild type. Seven hypervirulent mutants identified in this screen moved faster and reached higher populations than the wild type in grapevines. These results suggest that X. fastidosa attenuates its virulence in planta and that movement is important in X. fastidosa virulence. The mutated genes were sequenced and none had been described previously as antivirulence genes, although six of them showed similarity with genes of known functions in other organisms. One transposon insertion inactivated a hemagglutinin adhesin gene (PD2118), which we named HxfA. Another mutant in a second putative X. fastidosa hemagglutinin gene, PD1792 (HxfB), was constructed, and further characterization of these hxf mutants suggests that X. fastidosa hemagglutinins mediate contact between X. fastidosa cells, which results in colony formation and biofilm maturation within the xylem vessels.

  3. Structural insights into Rhino-Deadlock complex for germline piRNA cluster specification.

    Science.gov (United States)

    Yu, Bowen; Lin, Yu An; Parhad, Swapnil S; Jin, Zhaohui; Ma, Jinbiao; Theurkauf, William E; Zhang, Zz Zhao; Huang, Ying

    2018-06-01

    PIWI-interacting RNAs (piRNAs) silence transposons in germ cells to maintain genome stability and animal fertility. Rhino, a rapidly evolving heterochromatin protein 1 (HP1) family protein, binds Deadlock in a species-specific manner and so defines the piRNA-producing loci in the Drosophila genome. Here, we determine the crystal structures of Rhino-Deadlock complex in Drosophila melanogaster and simulans In both species, one Rhino binds the N-terminal helix-hairpin-helix motif of one Deadlock protein through a novel interface formed by the beta-sheet in the Rhino chromoshadow domain. Disrupting the interface leads to infertility and transposon hyperactivation in flies. Our structural and functional experiments indicate that electrostatic repulsion at the interaction interface causes cross-species incompatibility between the sibling species. By determining the molecular architecture of this piRNA-producing machinery, we discover a novel HP1-partner interacting mode that is crucial to piRNA biogenesis and transposon silencing. We thus explain the cross-species incompatibility of two sibling species at the molecular level. © 2018 The Authors.

  4. Characterization of a group of MITEs with unusual features from two coral genomes.

    Directory of Open Access Journals (Sweden)

    Shi Wang

    Full Text Available BACKGROUND: Miniature inverted-repeat transposable elements (MITEs, which are common in eukaryotic genomes, are small non-coding elements that transpose by utilizing transposases encoded by autonomous transposons. Recent genome-wide analyses and cross-mobilization assays have greatly improved our knowledge on MITE proliferation, however, specific mechanisms for the origin and evolution of MITEs are still unclear. PRINCIPAL FINDINGS: A group of coral MITEs called CMITE were identified from two corals, Acropora millepora and Acropora palmata. CMITEs conform to many common characteristics of MITEs, but also present several unusual features. The most unusual feature of CMITEs is conservation of the internal region, which is more conserved between MITE families than the TIRs. The origin of this internal region remains unknown, although we found one CMITE family that seems to be derived from a piggyBac-like transposon in A. millepora. CMITEs can form tandem arrays, suggesting an unconventional way for MITEs to increase copy numbers. We also describe a case in which a novel transposable element was created by a CMITE insertion event. CONCLUSIONS: To our knowledge, this is the first report of identification of MITEs from coral genomes. Proliferation of CMITEs seems to be related to the transposition machinery of piggyBac-like autonomous transposons. The highly conserved internal region of CMITEs suggests a potential role for this region in their successful transposition. However, the origin of these unusual features in CMITEs remains unclear, and thus represents an intriguing topic for future investigations.

  5. Characterization of a group of MITEs with unusual features from two coral genomes.

    Science.gov (United States)

    Wang, Shi; Zhang, Lingling; Meyer, Eli; Matz, Mikhail V

    2010-05-18

    Miniature inverted-repeat transposable elements (MITEs), which are common in eukaryotic genomes, are small non-coding elements that transpose by utilizing transposases encoded by autonomous transposons. Recent genome-wide analyses and cross-mobilization assays have greatly improved our knowledge on MITE proliferation, however, specific mechanisms for the origin and evolution of MITEs are still unclear. A group of coral MITEs called CMITE were identified from two corals, Acropora millepora and Acropora palmata. CMITEs conform to many common characteristics of MITEs, but also present several unusual features. The most unusual feature of CMITEs is conservation of the internal region, which is more conserved between MITE families than the TIRs. The origin of this internal region remains unknown, although we found one CMITE family that seems to be derived from a piggyBac-like transposon in A. millepora. CMITEs can form tandem arrays, suggesting an unconventional way for MITEs to increase copy numbers. We also describe a case in which a novel transposable element was created by a CMITE insertion event. To our knowledge, this is the first report of identification of MITEs from coral genomes. Proliferation of CMITEs seems to be related to the transposition machinery of piggyBac-like autonomous transposons. The highly conserved internal region of CMITEs suggests a potential role for this region in their successful transposition. However, the origin of these unusual features in CMITEs remains unclear, and thus represents an intriguing topic for future investigations.

  6. The complete and fully assembled genome sequence of Aeromonas salmonicida subsp. pectinolytica and its comparative analysis with other Aeromonas species: investigation of the mobilome in environmental and pathogenic strains.

    Science.gov (United States)

    Pfeiffer, Friedhelm; Zamora-Lagos, Maria-Antonia; Blettinger, Martin; Yeroslaviz, Assa; Dahl, Andreas; Gruber, Stephan; Habermann, Bianca H

    2018-01-05

    Due to the predominant usage of short-read sequencing to date, most bacterial genome sequences reported in the last years remain at the draft level. This precludes certain types of analyses, such as the in-depth analysis of genome plasticity. Here we report the finalized genome sequence of the environmental strain Aeromonas salmonicida subsp. pectinolytica 34mel, for which only a draft genome with 253 contigs is currently available. Successful completion of the transposon-rich genome critically depended on the PacBio long read sequencing technology. Using finalized genome sequences of A. salmonicida subsp. pectinolytica and other Aeromonads, we report the detailed analysis of the transposon composition of these bacterial species. Mobilome evolution is exemplified by a complex transposon, which has shifted from pathogenicity-related to environmental-related gene content in A. salmonicida subsp. pectinolytica 34mel. Obtaining the complete, circular genome of A. salmonicida subsp. pectinolytica allowed us to perform an in-depth analysis of its mobilome. We demonstrate the mobilome-dependent evolution of this strain's genetic profile from pathogenic to environmental.

  7. The Candidate Cancer Gene Database: a database of cancer driver genes from forward genetic screens in mice.

    Science.gov (United States)

    Abbott, Kenneth L; Nyre, Erik T; Abrahante, Juan; Ho, Yen-Yi; Isaksson Vogel, Rachel; Starr, Timothy K

    2015-01-01

    Identification of cancer driver gene mutations is crucial for advancing cancer therapeutics. Due to the overwhelming number of passenger mutations in the human tumor genome, it is difficult to pinpoint causative driver genes. Using transposon mutagenesis in mice many laboratories have conducted forward genetic screens and identified thousands of candidate driver genes that are highly relevant to human cancer. Unfortunately, this information is difficult to access and utilize because it is scattered across multiple publications using different mouse genome builds and strength metrics. To improve access to these findings and facilitate meta-analyses, we developed the Candidate Cancer Gene Database (CCGD, http://ccgd-starrlab.oit.umn.edu/). The CCGD is a manually curated database containing a unified description of all identified candidate driver genes and the genomic location of transposon common insertion sites (CISs) from all currently published transposon-based screens. To demonstrate relevance to human cancer, we performed a modified gene set enrichment analysis using KEGG pathways and show that human cancer pathways are highly enriched in the database. We also used hierarchical clustering to identify pathways enriched in blood cancers compared to solid cancers. The CCGD is a novel resource available to scientists interested in the identification of genetic drivers of cancer. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Candidate Essential Genes in Burkholderia cenocepacia J2315 Identified by Genome-Wide TraDIS

    KAUST Repository

    Wong, Yee-Chin; Abd El Ghany, Moataz; Naeem, Raeece; Lee, Kok-Wei; Tan, Yung-Chie; Pain, Arnab; Nathan, Sheila

    2016-01-01

    Burkholderia cenocepacia infection often leads to fatal cepacia syndrome in cystic fibrosis patients. However, antibiotic therapy rarely results in complete eradication of the pathogen due to its intrinsic resistance to many clinically available antibiotics. Recent attention has turned to the identification of essential genes as the proteins encoded by these genes may serve as potential targets for development of novel antimicrobials. In this study, we utilized TraDIS (Transposon Directed Insertion-site Sequencing) as a genome-wide screening tool to facilitate the identification of B. cenocepacia genes essential for its growth and viability. A transposon mutant pool consisting of approximately 500,000 mutants was successfully constructed, with more than 400,000 unique transposon insertion sites identified by computational analysis of TraDIS datasets. The saturated library allowed for the identification of 383 genes that were predicted to be essential in B. cenocepacia. We extended the application of TraDIS to identify conditionally essential genes required for in vitro growth and revealed an additional repertoire of 439 genes to be crucial for B. cenocepacia growth under nutrient-depleted conditions. The library of B. cenocepacia mutants can subsequently be subjected to various biologically related conditions to facilitate the discovery of genes involved in niche adaptation as well as pathogenicity and virulence.

  9. Seedling lethality in Nicotiana plumbaginifolia conferred by Ds transposable element insertion into a plant-specific gene.

    Science.gov (United States)

    Majira, Amel; Domin, Monique; Grandjean, Olivier; Gofron, Krystyna; Houba-Hérin, Nicole

    2002-10-01

    A seedling lethal mutant of Nicotiana plumbaginifolia (sdl-1) was isolated by transposon tagging using a maize Dissociation (Ds) element. The insertion mutation was produced by direct co-transformation of protoplasts with two plasmids: one containing Ds and a second with an Ac transposase gene. sdl-1 seedlings exhibit several phenotypes: swollen organs, short hypocotyls in light and dark conditions, and enlarged and multinucleated cells, that altogether suggest cell growth defects. Mutant cells are able to proliferate under in vitro culture conditions. Genomic DNA sequences bordering the transposon were used to recover cDNA from the normal allele. Complementation of the mutant phenotype with the cDNA confirmed that the transposon had caused the mutation. The Ds element was inserted into the first exon of the open reading frame and the homozygous mutant lacked detectable transcript. Phenocopies of the mutant were obtained by an antisense approach. SDL-1 encodes a novel protein found in several plant genomes but apparently missingfrom animal and fungal genomes; the protein is highly conserved and has a potential plastid targeting motif.

  10. Identification of a locus controlling expression of luminescence genes in Vibrio harveyi.

    Science.gov (United States)

    Martin, M; Showalter, R; Silverman, M

    1989-05-01

    Mutagenesis with transposon mini-Mulac was used to identify loci containing genes for bioluminescence (lux) in the marine bacterium Vibrio harveyi. Transposon insertions which resulted in a Lux- phenotype were mapped to two unlinked regions of the genome. Region I contained the luxCDABE operon which was previously shown to encode the enzymes luciferase and fatty acid reductase, which are required for light production. The other locus, region II, which was identified for the first time in this study, appeared to have a regulatory function. In Northern blot analysis of mRNA from mutants with defects in this region, no transcription from the luxCDABE operon could be detected. Strains with transposon-generated lux::lacZ gene fusions were used to analyze control of the transcription of these regions. Expression of luminescence in the wild type was strongly influenced by the density of the culture, and in strains with the lacZ indicator gene coupled to the luxCDABE operon, beta-galactosidase synthesis was density dependent. So, transcription of this operon is responsive to a density-sensing mechanism. However, beta-galactosidase synthesis in strains with lacZ fused to the region II transcriptional unit did not respond to cell density. The organization and regulation of the lux genes of V. harveyi are discussed, particularly with regard to the contrasts observed with the lux system of the fish light-organ symbiont Vibrio fischeri.

  11. Identification of Salmonella typhimurium Genes Required for Colonization of the Chicken Alimentary Tract and for Virulence in Newly Hatched Chicks

    Science.gov (United States)

    Turner, Arthur K.; Lovell, Margaret A.; Hulme, Scott D.; Zhang-Barber, Li; Barrow, Paul A.

    1998-01-01

    From a collection of 2,800 Tn5-TC1 transposon mutants of Salmonella typhimurium F98, 18 that showed reduced intestinal colonization of 3-week-old chicks were identified. The sites of transposon insertion were determined for most of the mutants and included insertions in the lipopolysaccharide biosynthesis genes rfaK, rfaY, rfbK, and rfbB and the genes dksA, clpB, hupA, and sipC. In addition, identification was made of an insertion into a novel gene that encodes a protein showing similarity to the IIC component of the mannose class of phosphoenolpyruvate-carbohydrate phosphotransferase systems, which we putatively called ptsC. Transduction of most of the transposon mutations to a fresh S. typhimurium F98 genetic background and construction of defined mutations in the rfbK, dksA, hupA, sipC, and ptsC genes of S. typhimurium F98 supported the role in colonization of all but the pts locus. The virulence of the rfbK, dksA, hupA, sipC, and ptsC defined mutants and clpB and rfaY transductants in 1-day-old chicks was tested. All but the ptsC and rfaY mutants were attenuated for virulence. A number of other phenotypes associated with some of the mutations are described. PMID:9573095

  12. Minotaur is critical for primary piRNA biogenesis

    Science.gov (United States)

    Vagin, Vasily V.; Yu, Yang; Jankowska, Anna; Luo, Yicheng; Wasik, Kaja A.; Malone, Colin D.; Harrison, Emily; Rosebrock, Adam; Wakimoto, Barbara T.; Fagegaltier, Delphine; Muerdter, Felix; Hannon, Gregory J.

    2013-01-01

    Piwi proteins and their associated small RNAs are essential for fertility in animals. In part, this is due to their roles in guarding germ cell genomes against the activity of mobile genetic elements. piRNA populations direct Piwi proteins to silence transposon targets and, as such, form a molecular code that discriminates transposons from endogenous genes. Information ultimately carried by piRNAs is encoded within genomic loci, termed piRNA clusters. These give rise to long, single-stranded, primary transcripts that are processed into piRNAs. Despite the biological importance of this pathway, neither the characteristics that define a locus as a source of piRNAs nor the mechanisms that catalyze primary piRNA biogenesis are well understood. We searched an EMS-mutant collection annotated for fertility phenotypes for genes involved in the piRNA pathway. Twenty-seven homozygous sterile strains showed transposon-silencing defects. One of these, which strongly impacted primary piRNA biogenesis, harbored a causal mutation in CG5508, a member of the Drosophila glycerol-3-phosphate O-acetyltransferase (GPAT) family. These enzymes catalyze the first acylation step on the path to the production of phosphatidic acid (PA). Though this pointed strongly to a function for phospholipid signaling in the piRNA pathway, a mutant form of CG5508, which lacks the GPAT active site, still functions in piRNA biogenesis. We have named this new biogenesis factor Minotaur. PMID:23788724

  13. Candidate essential genes in Burkholderia cenocepacia J2315 identified by genome-wide TraDIS

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    Yee-Chin Wong

    2016-08-01

    Full Text Available Burkholderia cenocepacia infection often leads to fatal cepacia syndrome in cystic fibrosis patients. However, antibiotic therapy rarely results in complete eradication of the pathogen due to its intrinsic resistance to many clinically available antibiotics. Recent attention has turned to the identification of essential genes as the proteins encoded by these genes may serve as potential targets for development of novel antimicrobials. In this study, we utilized TraDIS (Transposon Directed Insertion-site Sequencing as a genome-wide screening tool to facilitate the identification of B. cenocepacia genes essential for its growth and viability. A transposon mutant pool consisting of approximately 500,000 mutants was successfully constructed, with more than 400,000 unique transposon insertion sites identified by computational analysis of TraDIS datasets. The saturated library allowed for the identification of 383 genes that were predicted to be essential in B. cenocepacia. We extended the application of TraDIS to identify conditionally essential genes required for in vitro growth and revealed an additional repertoire of 439 genes to be crucial for B. cenocepacia growth under nutrient-depleted conditions. The library of B. cenocepacia mutants can subsequently be subjected to various biologically related conditions to facilitate the discovery of genes involved in niche adaptation as well as pathogenicity and virulence.

  14. Splinkerette PCR for mapping transposable elements in Drosophila.

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    Christopher J Potter

    2010-04-01

    Full Text Available Transposable elements (such as the P-element and piggyBac have been used to introduce thousands of transgenic constructs into the Drosophila genome. These transgenic constructs serve many roles, from assaying gene/cell function, to controlling chromosome arm rearrangement. Knowing the precise genomic insertion site for the transposable element is often desired. This enables identification of genomic enhancer regions trapped by an enhancer trap, identification of the gene mutated by a transposon insertion, or simplifying recombination experiments. The most commonly used transgene mapping method is inverse PCR (iPCR. Although usually effective, limitations with iPCR hinder its ability to isolate flanking genomic DNA in complex genomic loci, such as those that contain natural transposons. Here we report the adaptation of the splinkerette PCR (spPCR method for the isolation of flanking genomic DNA of any P-element or piggyBac. We report a simple and detailed protocol for spPCR. We use spPCR to 1 map a GAL4 enhancer trap located inside a natural transposon, pinpointing a master regulatory region for olfactory neuron expression in the brain; and 2 map all commonly used centromeric FRT insertion sites. The ease, efficiency, and efficacy of spPCR could make it a favored choice for the mapping of transposable element in Drosophila.

  15. Universal platform for quantitative analysis of DNA transposition

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    Pajunen Maria I

    2010-11-01

    Full Text Available Abstract Background Completed genome projects have revealed an astonishing diversity of transposable genetic elements, implying the existence of novel element families yet to be discovered from diverse life forms. Concurrently, several better understood transposon systems have been exploited as efficient tools in molecular biology and genomics applications. Characterization of new mobile elements and improvement of the existing transposition technology platforms warrant easy-to-use assays for the quantitative analysis of DNA transposition. Results Here we developed a universal in vivo platform for the analysis of transposition frequency with class II mobile elements, i.e., DNA transposons. For each particular transposon system, cloning of the transposon ends and the cognate transposase gene, in three consecutive steps, generates a multifunctional plasmid, which drives inducible expression of the transposase gene and includes a mobilisable lacZ-containing reporter transposon. The assay scores transposition events as blue microcolonies, papillae, growing within otherwise whitish Escherichia coli colonies on indicator plates. We developed the assay using phage Mu transposition as a test model and validated the platform using various MuA transposase mutants. For further validation and to illustrate universality, we introduced IS903 transposition system components into the assay. The developed assay is adjustable to a desired level of initial transposition via the control of a plasmid-borne E. coli arabinose promoter. In practice, the transposition frequency is modulated by varying the concentration of arabinose or glucose in the growth medium. We show that variable levels of transpositional activity can be analysed, thus enabling straightforward screens for hyper- or hypoactive transposase mutants, regardless of the original wild-type activity level. Conclusions The established universal papillation assay platform should be widely applicable to a

  16. Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana

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    You Wanhui

    2012-04-01

    Full Text Available Abstract Background In plants, transposons and non-protein-coding repeats are epigenetically silenced by CG and non-CG methylation. This pattern of methylation is mediated in part by small RNAs and two specialized RNA polymerases, termed Pol IV and Pol V, in a process called RNA-directed DNA methylation. By contrast, many protein-coding genes transcribed by Pol II contain in their gene bodies exclusively CG methylation that is independent of small RNAs and Pol IV/Pol V activities. It is unclear how the different methylation machineries distinguish between transposons and genes. Here we report on a group of atypical genes that display in their coding region a transposon-like methylation pattern, which is associated with gene silencing in sporophytic tissues. Results We performed a methylation-sensitive amplification polymorphism analysis to search for targets of RNA-directed DNA methylation in Arabidopsis thaliana and identified several members of a gene family encoding cysteine-rich peptides (CRPs. In leaves, the CRP genes are silent and their coding regions contain dense, transposon-like methylation in CG, CHG and CHH contexts, which depends partly on the Pol IV/Pol V pathway and small RNAs. Methylation in the coding region is reduced, however, in the synergid cells of the female gametophyte, where the CRP genes are specifically expressed. Further demonstrating that expressed CRP genes lack gene body methylation, a CRP4-GFP fusion gene under the control of the constitutive 35 S promoter remains unmethylated in leaves and is transcribed to produce a translatable mRNA. By contrast, a CRP4-GFP fusion gene under the control of a CRP4 promoter fragment acquires CG and non-CG methylation in the CRP coding region in leaves similar to the silent endogenous CRP4 gene. Conclusions Unlike CG methylation in gene bodies, which does not dramatically affect Pol II transcription, combined CG and non-CG methylation in CRP coding regions is likely to

  17. Mobile genetic elements, a key to microbial adaptation in extreme environments

    Science.gov (United States)

    van Houdt, Rob; Mijnendonckx, Kristel; Provoost, Ann; Monsieurs, Pieter; Mergeay, Max; Leys, Natalie

    hydrogenotrophy. Five transposons were identified in CH34. Two mercury transposons Tn4378 and Tn4380, respectively located on pMOL28 and pMOL30, were previously described. In addition, 3 novel transposons were identified. The large Tn6048 transposon contained 8 genes highly induced by zinc and lead. Transposon Tn6049 was found in twelve copies in the genome of strain CH34 and is seem-ingly often associated with genomic islands. Finally, Tn6050 was observed twice in the second chromosome with accessory genes not classically associated with transposons, namely a sulfate permease, a universal stress protein (UspA) and a DksA-like DnaK suppressor protein. The role of these genes is unclear but a functional association could be assumed. Finally, a set of 21 IS elements was found, counting in total for 57 copies dispersed over the genome. The number of copies ranged from 1 to 9 (IS1088 ) and 10 (ISRme3 ). The 21 IS elements could be divided into 10 different families. The elements ISRme5 and IS1071 were putatively involved in the recruitment of the genes for hydrogenotrophy and CO2 fixation, with mutants unable to grow on H2 and CO2 appeared to have lost these genes by IS1071 -mediated excision. These data clearly showed that the C. metallidurans CH34 model bacterium and some of it's Cupriavidus and Ralstonia relatives carry a multitude of tools that allowed them to genetically adapt to polluted and extreme soil environments, and that are a key in their success to adapt to the new anthropogenic spacecraft environments. Furthermore, these tools could be exploited to assess and measure genetic effects of spaceflight conditions. Acknowledgements This work was supported by the European Space Agency (ESA-PRODEX) and the Belgian Science Policy (Belspo) through the MISSEX and COMICS projects.

  18. Characterization of extended-spectrum β-lactamases (ESBLs)-producing Salmonella in retail raw chicken carcasses.

    Science.gov (United States)

    Qiao, Jing; Zhang, Qiang; Alali, Walid Q; Wang, Jiawei; Meng, Lingyuan; Xiao, Yingping; Yang, Hua; Chen, Sheng; Cui, Shenghui; Yang, Baowei

    2017-05-02

    Extended-spectrum β-lactamases (ESBLs)-producing Salmonella is considered a serious concern to public health worldwide. However, limited information is available on ESBLs-producing Salmonella in retail chicken products in China. The objective of this study was to characterize ESBLs-producing Salmonella isolates from retail chickens in China. A total of 890 Salmonella isolates from retail chicken carcasses collected from 4 provinces were firstly screened for ESBLs-production phenotype via the double-disk synergy test method. A total of 96 (10.8%, n=890) ESBLs-producing Salmonella were identified and subjected to PFGE analysis, characterization for the presence of ESBLs encoding genes, transposons, carbapenemase and virulence genes. A total of 59 PFGE profiles were detected in these 96 isolates, among which 57.3% were found to harbor bla TEM-1 , whereas 30.2%, 24.0%, 18.8% and 7.3% were carrying bla OXA-1 , bla CTX-M-15 , bla CTX-M-3 and bla PSE-1 genes, respectively. Moreover, 42 (43.8%) isolates co-carried 2 ESBLs-producing genes, and two (2.1%) isolates co-carried 3 genes. Furthermore, 24 (25.0%) ESBLs-producing isolates carried VIM and 10 (10.4%) carried KPC encoding genes that closely associated with carbapenems resistance. Eighty-eight isolates harbored transposons ranging from 4.2% for Tn903 to 76.0% for Tn21. Out of the 88 Salmonella that harbored transposons, 25%, 22.7%, 23.9%, 10.2% and 1.1% of isolates were found to carry 2, 3, 4, 5 and 6 transposons, respectively. The minimum inhibitory concentration (MIC) values for cephalosporins (ceftriaxone, cefoperazone and cefoxitin) to ESBLs-producing isolates were from 4 to 1024μg/mL, for nalidixic acid were from 64 to 512μg/mL, for fluoroquinolones (ciprofloxacin, levofloxacin and gatifloxacin) were from 4 to 256μg/mL. Twenty-nine virulence genes were detected in the 96 ESBLs-producing isolates with 2.1% harbored spvR (lowest) and 90.6% harbored marT and steB (highest). All isolates carried at least one

  19. Regulation of mariner transposition: the peculiar case of Mos1.

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    Jérôme Jaillet

    Full Text Available BACKGROUND: Mariner elements represent the most successful family of autonomous DNA transposons, being present in various plant and animal genomes, including humans. The introduction and co-evolution of mariners within host genomes imply a strict regulation of the transposon activity. Biochemical data accumulated during the past decade have led to a convergent picture of the transposition cycle of mariner elements, suggesting that mariner transposition does not rely on host-specific factors. This model does not account for differences of transposition efficiency in human cells between mariners. We thus wondered whether apparent similarities in transposition cycle could hide differences in the intrinsic parameters that control mariner transposition. PRINCIPAL FINDINGS: We find that Mos1 transposase concentrations in excess to the Mos1 ends prevent the paired-end complex assembly. However, we observe that Mos1 transposition is not impaired by transposase high concentration, dismissing the idea that transposase over production plays an obligatory role in the down-regulation of mariner transposition. Our main finding is that the paired-end complex is formed in a cooperative way, regardless of the transposase concentration. We also show that an element framed by two identical ITRs (Inverted Terminal Repeats is more efficient in driving transposition than an element framed by two different ITRs (i.e. the natural Mos1 copy, the latter being more sensitive to transposase concentration variations. Finally, we show that the current Mos1 ITRs correspond to the ancestral ones. CONCLUSIONS: We provide new insights on intrinsic properties supporting the self-regulation of the Mos1 element. These properties (transposase specific activity, aggregation, ITR sequences, transposase concentration/transposon copy number ratio... could have played a role in the dynamics of host-genomes invasion by Mos1, accounting (at least in part for the current low copy number of

  20. Mycothiol-Deficient Mycobacterium smegmatis Mutants Are Hypersensitive to Alkylating Agents, Free Radicals, and Antibiotics

    Science.gov (United States)

    Rawat, Mamta; Newton, Gerald L.; Ko, Mary; Martinez, Gladys J.; Fahey, Robert C.; Av-Gay, Yossef

    2002-01-01

    Mycothiol (MSH; 1d-myo-inosityl 2-[N-acetyl-l-cysteinyl]amido-2-deoxy-α-d-glucopyranoside) is the major low-molecular-weight thiol produced by mycobacteria. Mutants of Mycobacterium smegmatis mc2155 deficient in MSH production were produced by chemical mutagenesis as well as by transposon mutagenesis. One chemical mutant (mutant I64) and two transposon mutants (mutants Tn1 and Tn2) stably deficient in MSH production were isolated by screening for reduced levels of MSH content. The MSH contents of transposon mutants Tn1 and Tn2 were found to be less than 0.1% that of the parent strain, and the MSH content of I64 was found to be 1 to 5% that of the parent strain. All three strains accumulated 1d-myo-inosityl 2-deoxy-α-d-glucopyranoside to levels 20- to 25-fold the level found in the parent strain. The cysteine:1d-myo-inosityl 2-amino-2-deoxy-α-d-glucopyranoside ligase (MshC) activities of the three mutant strains were ≤2% that of the parent strain. Phenotypic analysis revealed that these MSH-deficient mutants possess increased susceptibilities to free radicals and alkylating agents and to a wide range of antibiotics including erythromycin, azithromycin, vancomycin, penicillin G, rifamycin, and rifampin. Conversely, the mutants possess at least 200-fold higher levels of resistance to isoniazid than the wild type. We mapped the mutation in the chemical mutant by sequencing the mshC gene and showed that a single amino acid substitution (L205P) is responsible for reduced MSH production and its associated phenotype. Our results demonstrate that there is a direct correlation between MSH depletion and enhanced sensitivity to toxins and antibiotics. PMID:12384335

  1. Sigma E regulators control hemolytic activity and virulence in a shrimp pathogenic Vibrio harveyi.

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    Pimonsri Rattanama

    Full Text Available Members of the genus Vibrio are important marine and aquaculture pathogens. Hemolytic activity has been identified as a virulence factor in many pathogenic vibrios including V. cholerae, V. parahaemolyticus, V. alginolyticus, V. harveyi and V. vulnificus. We have used transposon mutagenesis to identify genes involved in the hemolytic activity of shrimp-pathogenic V. harveyi strain PSU3316. Out of 1,764 mutants screened, five mutants showed reduced hemolytic activity on sheep blood agar and exhibited virulence attenuation in shrimp (Litopenaeus vannamei. Mutants were identified by comparing transposon junction sequences to a draft of assembly of the PSU3316 genome. Surprisingly none of the disrupted open reading frames or gene neighborhoods contained genes annotated as hemolysins. The gene encoding RseB, a negative regulator of the sigma factor (σ(E, was interrupted in 2 out of 5 transposon mutants, in addition, the transcription factor CytR, a threonine synthetase, and an efflux-associated cytoplasmic protein were also identified. Knockout mutations introduced into the rpoE operon at the rseB gene exhibited low hemolytic activity in sheep blood agar, and were 3-to 7-fold attenuated for colonization in shrimp. Comparison of whole cell extracted proteins in the rseB mutant (PSU4030 to the wild-type by 2-D gel electrophoresis revealed 6 differentially expressed proteins, including two down-regulated porins (OmpC-like and OmpN and an upregulated protease (DegQ which have been associated with σ(E in other organisms. Our study is the first report linking hemolytic activity to the σ(E regulators in pathogenic Vibrio species and suggests expression of this virulence-linked phenotype is governed by multiple regulatory pathways within the V. harveyi.

  2. Drosophila Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution

    Science.gov (United States)

    Leung, Wilson; Shaffer, Christopher D.; Reed, Laura K.; Smith, Sheryl T.; Barshop, William; Dirkes, William; Dothager, Matthew; Lee, Paul; Wong, Jeannette; Xiong, David; Yuan, Han; Bedard, James E. J.; Machone, Joshua F.; Patterson, Seantay D.; Price, Amber L.; Turner, Bryce A.; Robic, Srebrenka; Luippold, Erin K.; McCartha, Shannon R.; Walji, Tezin A.; Walker, Chelsea A.; Saville, Kenneth; Abrams, Marita K.; Armstrong, Andrew R.; Armstrong, William; Bailey, Robert J.; Barberi, Chelsea R.; Beck, Lauren R.; Blaker, Amanda L.; Blunden, Christopher E.; Brand, Jordan P.; Brock, Ethan J.; Brooks, Dana W.; Brown, Marie; Butzler, Sarah C.; Clark, Eric M.; Clark, Nicole B.; Collins, Ashley A.; Cotteleer, Rebecca J.; Cullimore, Peterson R.; Dawson, Seth G.; Docking, Carter T.; Dorsett, Sasha L.; Dougherty, Grace A.; Downey, Kaitlyn A.; Drake, Andrew P.; Earl, Erica K.; Floyd, Trevor G.; Forsyth, Joshua D.; Foust, Jonathan D.; Franchi, Spencer L.; Geary, James F.; Hanson, Cynthia K.; Harding, Taylor S.; Harris, Cameron B.; Heckman, Jonathan M.; Holderness, Heather L.; Howey, Nicole A.; Jacobs, Dontae A.; Jewell, Elizabeth S.; Kaisler, Maria; Karaska, Elizabeth A.; Kehoe, James L.; Koaches, Hannah C.; Koehler, Jessica; Koenig, Dana; Kujawski, Alexander J.; Kus, Jordan E.; Lammers, Jennifer A.; Leads, Rachel R.; Leatherman, Emily C.; Lippert, Rachel N.; Messenger, Gregory S.; Morrow, Adam T.; Newcomb, Victoria; Plasman, Haley J.; Potocny, Stephanie J.; Powers, Michelle K.; Reem, Rachel M.; Rennhack, Jonathan P.; Reynolds, Katherine R.; Reynolds, Lyndsey A.; Rhee, Dong K.; Rivard, Allyson B.; Ronk, Adam J.; Rooney, Meghan B.; Rubin, Lainey S.; Salbert, Luke R.; Saluja, Rasleen K.; Schauder, Taylor; Schneiter, Allison R.; Schulz, Robert W.; Smith, Karl E.; Spencer, Sarah; Swanson, Bryant R.; Tache, Melissa A.; Tewilliager, Ashley A.; Tilot, Amanda K.; VanEck, Eve; Villerot, Matthew M.; Vylonis, Megan B.; Watson, David T.; Wurzler, Juliana A.; Wysocki, Lauren M.; Yalamanchili, Monica; Zaborowicz, Matthew A.; Emerson, Julia A.; Ortiz, Carlos; Deuschle, Frederic J.; DiLorenzo, Lauren A.; Goeller, Katie L.; Macchi, Christopher R.; Muller, Sarah E.; Pasierb, Brittany D.; Sable, Joseph E.; Tucci, Jessica M.; Tynon, Marykathryn; Dunbar, David A.; Beken, Levent H.; Conturso, Alaina C.; Danner, Benjamin L.; DeMichele, Gabriella A.; Gonzales, Justin A.; Hammond, Maureen S.; Kelley, Colleen V.; Kelly, Elisabeth A.; Kulich, Danielle; Mageeney, Catherine M.; McCabe, Nikie L.; Newman, Alyssa M.; Spaeder, Lindsay A.; Tumminello, Richard A.; Revie, Dennis; Benson, Jonathon M.; Cristostomo, Michael C.; DaSilva, Paolo A.; Harker, Katherine S.; Jarrell, Jenifer N.; Jimenez, Luis A.; Katz, Brandon M.; Kennedy, William R.; Kolibas, Kimberly S.; LeBlanc, Mark T.; Nguyen, Trung T.; Nicolas, Daniel S.; Patao, Melissa D.; Patao, Shane M.; Rupley, Bryan J.; Sessions, Bridget J.; Weaver, Jennifer A.; Goodman, Anya L.; Alvendia, Erica L.; Baldassari, Shana M.; Brown, Ashley S.; Chase, Ian O.; Chen, Maida; Chiang, Scott; Cromwell, Avery B.; Custer, Ashley F.; DiTommaso, Tia M.; El-Adaimi, Jad; Goscinski, Nora C.; Grove, Ryan A.; Gutierrez, Nestor; Harnoto, Raechel S.; Hedeen, Heather; Hong, Emily L.; Hopkins, Barbara L.; Huerta, Vilma F.; Khoshabian, Colin; LaForge, Kristin M.; Lee, Cassidy T.; Lewis, Benjamin M.; Lydon, Anniken M.; Maniaci, Brian J.; Mitchell, Ryan D.; Morlock, Elaine V.; Morris, William M.; Naik, Priyanka; Olson, Nicole C.; Osterloh, Jeannette M.; Perez, Marcos A.; Presley, Jonathan D.; Randazzo, Matt J.; Regan, Melanie K.; Rossi, Franca G.; Smith, Melanie A.; Soliterman, Eugenia A.; Sparks, Ciani J.; Tran, Danny L.; Wan, Tiffany; Welker, Anne A.; Wong, Jeremy N.; Sreenivasan, Aparna; Youngblom, Jim; Adams, Andrew; Alldredge, Justin; Bryant, Ashley; Carranza, David; Cifelli, Alyssa; Coulson, Kevin; Debow, Calise; Delacruz, Noelle; Emerson, Charlene; Farrar, Cassandra; Foret, Don; Garibay, Edgar; Gooch, John; Heslop, Michelle; Kaur, Sukhjit; Khan, Ambreen; Kim, Van; Lamb, Travis; Lindbeck, Peter; Lucas, Gabi; Macias, Elizabeth; Martiniuc, Daniela; Mayorga, Lissett; Medina, Joseph; Membreno, Nelson; Messiah, Shady; Neufeld, Lacey; Nguyen, San Francisco; Nichols, Zachary; Odisho, George; Peterson, Daymon; Rodela, Laura; Rodriguez, Priscilla; Rodriguez, Vanessa; Ruiz, Jorge; Sherrill, Will; Silva, Valeria; Sparks, Jeri; Statton, Geeta; Townsend, Ashley; Valdez, Isabel; Waters, Mary; Westphal, Kyle; Winkler, Stacey; Zumkehr, Joannee; DeJong, Randall J.; Hoogewerf, Arlene J.; Ackerman, Cheri M.; Armistead, Isaac O.; Baatenburg, Lara; Borr, Matthew J.; Brouwer, Lindsay K.; Burkhart, Brandon J.; Bushhouse, Kelsey T.; Cesko, Lejla; Choi, Tiffany Y. Y.; Cohen, Heather; Damsteegt, Amanda M.; Darusz, Jess M.; Dauphin, Cory M.; Davis, Yelena P.; Diekema, Emily J.; Drewry, Melissa; Eisen, Michelle E. M.; Faber, Hayley M.; Faber, Katherine J.; Feenstra, Elizabeth; Felzer-Kim, Isabella T.; Hammond, Brandy L.; Hendriksma, Jesse; Herrold, Milton R.; Hilbrands, Julia A.; Howell, Emily J.; Jelgerhuis, Sarah A.; Jelsema, Timothy R.; Johnson, Benjamin K.; Jones, Kelly K.; Kim, Anna; Kooienga, Ross D.; Menyes, Erika E.; Nollet, Eric A.; Plescher, Brittany E.; Rios, Lindsay; Rose, Jenny L.; Schepers, Allison J.; Scott, Geoff; Smith, Joshua R.; Sterling, Allison M.; Tenney, Jenna C.; Uitvlugt, Chris; VanDyken, Rachel E.; VanderVennen, Marielle; Vue, Samantha; Kokan, Nighat P.; Agbley, Kwabea; Boham, Sampson K.; Broomfield, Daniel; Chapman, Kayla; Dobbe, Ali; Dobbe, Ian; Harrington, William; Ibrahem, Marwan; Kennedy, Andre; Koplinsky, Chad A.; Kubricky, Cassandra; Ladzekpo, Danielle; Pattison, Claire; Ramirez, Roman E.; Wande, Lucia; Woehlke, Sarah; Wawersik, Matthew; Kiernan, Elizabeth; Thompson, Jeffrey S.; Banker, Roxanne; Bartling, Justina R.; Bhatiya, Chinmoy I.; Boudoures, Anna L.; Christiansen, Lena; Fosselman, Daniel S.; French, Kristin M.; Gill, Ishwar S.; Havill, Jessen T.; Johnson, Jaelyn L.; Keny, Lauren J.; Kerber, John M.; Klett, Bethany M.; Kufel, Christina N.; May, Francis J.; Mecoli, Jonathan P.; Merry, Callie R.; Meyer, Lauren R.; Miller, Emily G.; Mullen, Gregory J.; Palozola, Katherine C.; Pfeil, Jacob J.; Thomas, Jessica G.; Verbofsky, Evan M.; Spana, Eric P.; Agarwalla, Anant; Chapman, Julia; Chlebina, Ben; Chong, Insun; Falk, I.N.; Fitzgibbons, John D.; Friedman, Harrison; Ighile, Osagie; Kim, Andrew J.; Knouse, Kristin A.; Kung, Faith; Mammo, Danny; Ng, Chun Leung; Nikam, Vinayak S.; Norton, Diana; Pham, Philip; Polk, Jessica W.; Prasad, Shreya; Rankin, Helen; Ratliff, Camille D.; Scala, Victoria; Schwartz, Nicholas U.; Shuen, Jessica A.; Xu, Amy; Xu, Thomas Q.; Zhang, Yi; Rosenwald, Anne G.; Burg, Martin G.; Adams, Stephanie J.; Baker, Morgan; Botsford, Bobbi; Brinkley, Briana; Brown, Carter; Emiah, Shadie; Enoch, Erica; Gier, Chad; Greenwell, Alyson; Hoogenboom, Lindsay; Matthews, Jordan E.; McDonald, Mitchell; Mercer, Amanda; Monsma, Nicholaus; Ostby, Kristine; Ramic, Alen; Shallman, Devon; Simon, Matthew; Spencer, Eric; Tomkins, Trisha; Wendland, Pete; Wylie, Anna; Wolyniak, Michael J.; Robertson, Gregory M.; Smith, Samuel I.; DiAngelo, Justin R.; Sassu, Eric D.; Bhalla, Satish C.; Sharif, Karim A.; Choeying, Tenzin; Macias, Jason S.; Sanusi, Fareed; Torchon, Karvyn; Bednarski, April E.; Alvarez, Consuelo J.; Davis, Kristen C.; Dunham, Carrie A.; Grantham, Alaina J.; Hare, Amber N.; Schottler, Jennifer; Scott, Zackary W.; Kuleck, Gary A.; Yu, Nicole S.; Kaehler, Marian M.; Jipp, Jacob; Overvoorde, Paul J.; Shoop, Elizabeth; Cyrankowski, Olivia; Hoover, Betsy; Kusner, Matt; Lin, Devry; Martinov, Tijana; Misch, Jonathan; Salzman, Garrett; Schiedermayer, Holly; Snavely, Michael; Zarrasola, Stephanie; Parrish, Susan; Baker, Atlee; Beckett, Alissa; Belella, Carissa; Bryant, Julie; Conrad, Turner; Fearnow, Adam; Gomez, Carolina; Herbstsomer, Robert A.; Hirsch, Sarah; Johnson, Christen; Jones, Melissa; Kabaso, Rita; Lemmon, Eric; Vieira, Carolina Marques dos Santos; McFarland, Darryl; McLaughlin, Christopher; Morgan, Abbie; Musokotwane, Sepo; Neutzling, William; Nietmann, Jana; Paluskievicz, Christina; Penn, Jessica; Peoples, Emily; Pozmanter, Caitlin; Reed, Emily; Rigby, Nichole; Schmidt, Lasse; Shelton, Micah; Shuford, Rebecca; Tirasawasdichai, Tiara; Undem, Blair; Urick, Damian; Vondy, Kayla; Yarrington, Bryan; Eckdahl, Todd T.; Poet, Jeffrey L.; Allen, Alica B.; Anderson, John E.; Barnett, Jason M.; Baumgardner, Jordan S.; Brown, Adam D.; Carney, Jordan E.; Chavez, Ramiro A.; Christgen, Shelbi L.; Christie, Jordan S.; Clary, Andrea N.; Conn, Michel A.; Cooper, Kristen M.; Crowley, Matt J.; Crowley, Samuel T.; Doty, Jennifer S.; Dow, Brian A.; Edwards, Curtis R.; Elder, Darcie D.; Fanning, John P.; Janssen, Bridget M.; Lambright, Anthony K.; Lane, Curtiss E.; Limle, Austin B.; Mazur, Tammy; McCracken, Marly R.; McDonough, Alexa M.; Melton, Amy D.; Minnick, Phillip J.; Musick, Adam E.; Newhart, William H.; Noynaert, Joseph W.; Ogden, Bradley J.; Sandusky, Michael W.; Schmuecker, Samantha M.; Shipman, Anna L.; Smith, Anna L.; Thomsen, Kristen M.; Unzicker, Matthew R.; Vernon, William B.; Winn, Wesley W.; Woyski, Dustin S.; Zhu, Xiao; Du, Chunguang; Ament, Caitlin; Aso, Soham; Bisogno, Laura Simone; Caronna, Jason; Fefelova, Nadezhda; Lopez, Lenin; Malkowitz, Lorraine; Marra, Jonathan; Menillo, Daniella; Obiorah, Ifeanyi; Onsarigo, Eric Nyabeta; Primus, Shekerah; Soos, Mahdi; Tare, Archana; Zidan, Ameer; Jones, Christopher J.; Aronhalt, Todd; Bellush, James M.; Burke, Christa; DeFazio, Steve; Does, Benjamin R.; Johnson, Todd D.; Keysock, Nicholas; Knudsen, Nelson H.; Messler, James; Myirski, Kevin; Rekai, Jade Lea; Rempe, Ryan Michael; Salgado, Michael S.; Stagaard, Erica; Starcher, Justin R.; Waggoner, Andrew W.; Yemelyanova, Anastasia K.; Hark, Amy T.; Bertolet, Anne; Kuschner, Cyrus E.; Parry, Kesley; Quach, Michael; Shantzer, Lindsey; Shaw, Mary E.; Smith, Mary A.; Glenn, Omolara; Mason, Portia; Williams, Charlotte; Key, S. Catherine Silver; Henry, Tyneshia C. P.; Johnson, Ashlee G.; White, Jackie X.; Haberman, Adam; Asinof, Sam; Drumm, Kelly; Freeburg, Trip; Safa, Nadia; Schultz, Darrin; Shevin, Yakov; Svoronos, Petros; Vuong, Tam; Wellinghoff, Jules; Hoopes, Laura L. M.; Chau, Kim M.; Ward, Alyssa; Regisford, E. Gloria C.; Augustine, LaJerald; Davis-Reyes, Brionna; Echendu, Vivienne; Hales, Jasmine; Ibarra, Sharon; Johnson, Lauriaun; Ovu, Steven; Braverman, John M.; Bahr, Thomas J.; Caesar, Nicole M.; Campana, Christopher; Cassidy, Daniel W.; Cognetti, Peter A.; English, Johnathan D.; Fadus, Matthew C.; Fick, Cameron N.; Freda, Philip J.; Hennessy, Bryan M.; Hockenberger, Kelsey; Jones, Jennifer K.; King, Jessica E.; Knob, Christopher R.; Kraftmann, Karen J.; Li, Linghui; Lupey, Lena N.; Minniti, Carl J.; Minton, Thomas F.; Moran, Joseph V.; Mudumbi, Krishna; Nordman, Elizabeth C.; Puetz, William J.; Robinson, Lauren M.; Rose, Thomas J.; Sweeney, Edward P.; Timko, Ashley S.; Paetkau, Don W.; Eisler, Heather L.; Aldrup, Megan E.; Bodenberg, Jessica M.; Cole, Mara G.; Deranek, Kelly M.; DeShetler, Megan; Dowd, Rose M.; Eckardt, Alexandra K.; Ehret, Sharon C.; Fese, Jessica; Garrett, Amanda D.; Kammrath, Anna; Kappes, Michelle L.; Light, Morgan R.; Meier, Anne C.; O’Rouke, Allison; Perella, Mallory; Ramsey, Kimberley; Ramthun, Jennifer R.; Reilly, Mary T.; Robinett, Deirdre; Rossi, Nadine L.; Schueler, Mary Grace; Shoemaker, Emma; Starkey, Kristin M.; Vetor, Ashley; Vrable, Abby; Chandrasekaran, Vidya; Beck, Christopher; Hatfield, Kristen R.; Herrick, Douglas A.; Khoury, Christopher B.; Lea, Charlotte; Louie, Christopher A.; Lowell, Shannon M.; Reynolds, Thomas J.; Schibler, Jeanine; Scoma, Alexandra H.; Smith-Gee, Maxwell T.; Tuberty, Sarah; Smith, Christopher D.; Lopilato, Jane E.; Hauke, Jeanette; Roecklein-Canfield, Jennifer A.; Corrielus, Maureen; Gilman, Hannah; Intriago, Stephanie; Maffa, Amanda; Rauf, Sabya A.; Thistle, Katrina; Trieu, Melissa; Winters, Jenifer; Yang, Bib; Hauser, Charles R.; Abusheikh, Tariq; Ashrawi, Yara; Benitez, Pedro; Boudreaux, Lauren R.; Bourland, Megan; Chavez, Miranda; Cruz, Samantha; Elliott, GiNell; Farek, Jesse R.; Flohr, Sarah; Flores, Amanda H.; Friedrichs, Chelsey; Fusco, Zach; Goodwin, Zane; Helmreich, Eric; Kiley, John; Knepper, John Mark; Langner, Christine; Martinez, Megan; Mendoza, Carlos; Naik, Monal; Ochoa, Andrea; Ragland, Nicolas; Raimey, England; Rathore, Sunil; Reza, Evangelina; Sadovsky, Griffin; Seydoux, Marie-Isabelle B.; Smith, Jonathan E.; Unruh, Anna K.; Velasquez, Vicente; Wolski, Matthew W.; Gosser, Yuying; Govind, Shubha; Clarke-Medley, Nicole; Guadron, Leslie; Lau, Dawn; Lu, Alvin; Mazzeo, Cheryl; Meghdari, Mariam; Ng, Simon; Pamnani, Brad; Plante, Olivia; Shum, Yuki Kwan Wa; Song, Roy; Johnson, Diana E.; Abdelnabi, Mai; Archambault, Alexi; Chamma, Norma; Gaur, Shailly; Hammett, Deborah; Kandahari, Adrese; Khayrullina, Guzal; Kumar, Sonali; Lawrence, Samantha; Madden, Nigel; Mandelbaum, Max; Milnthorp, Heather; Mohini, Shiv; Patel, Roshni; Peacock, Sarah J.; Perling, Emily; Quintana, Amber; Rahimi, Michael; Ramirez, Kristen; Singhal, Rishi; Weeks, Corinne; Wong, Tiffany; Gillis, Aubree T.; Moore, Zachary D.; Savell, Christopher D.; Watson, Reece; Mel, Stephanie F.; Anilkumar, Arjun A.; Bilinski, Paul; Castillo, Rostislav; Closser, Michael; Cruz, Nathalia M.; Dai, Tiffany; Garbagnati, Giancarlo F.; Horton, Lanor S.; Kim, Dongyeon; Lau, Joyce H.; Liu, James Z.; Mach, Sandy D.; Phan, Thu A.; Ren, Yi; Stapleton, Kenneth E.; Strelitz, Jean M.; Sunjed, Ray; Stamm, Joyce; Anderson, Morgan C.; Bonifield, Bethany Grace; Coomes, Daniel; Dillman, Adam; Durchholz, Elaine J.; Fafara-Thompson, Antoinette E.; Gross, Meleah J.; Gygi, Amber M.; Jackson, Lesley E.; Johnson, Amy; Kocsisova, Zuzana; Manghelli, Joshua L.; McNeil, Kylie; Murillo, Michael; Naylor, Kierstin L.; Neely, Jessica; Ogawa, Emmy E.; Rich, Ashley; Rogers, Anna; Spencer, J. Devin; Stemler, Kristina M.; Throm, Allison A.; Van Camp, Matt; Weihbrecht, Katie; Wiles, T. Aaron; Williams, Mallory A.; Williams, Matthew; Zoll, Kyle; Bailey, Cheryl; Zhou, Leming; Balthaser, Darla M.; Bashiri, Azita; Bower, Mindy E.; Florian, Kayla A.; Ghavam, Nazanin; Greiner-Sosanko, Elizabeth S.; Karim, Helmet; Mullen, Victor W.; Pelchen, Carly E.; Yenerall, Paul M.; Zhang, Jiayu; Rubin, Michael R.; Arias-Mejias, Suzette M.; Bermudez-Capo, Armando G.; Bernal-Vega, Gabriela V.; Colon-Vazquez, Mariela; Flores-Vazquez, Arelys; Gines-Rosario, Mariela; Llavona-Cartagena, Ivan G.; Martinez-Rodriguez, Javier O.; Ortiz-Fuentes, Lionel; Perez-Colomba, Eliezer O.; Perez-Otero, Joseph; Rivera, Elisandra; Rodriguez-Giron, Luke J.; Santiago-Sanabria, Arnaldo J.; Senquiz-Gonzalez, Andrea M.; delValle, Frank R. Soto; Vargas-Franco, Dorianmarie; Velázquez-Soto, Karla I.; Zambrana-Burgos, Joan D.; Martinez-Cruzado, Juan Carlos; Asencio-Zayas, Lillyann; Babilonia-Figueroa, Kevin; Beauchamp-Pérez, Francis D.; Belén-Rodríguez, Juliana; Bracero-Quiñones, Luciann; Burgos-Bula, Andrea P.; Collado-Méndez, Xavier A.; Colón-Cruz, Luis R.; Correa-Muller, Ana I.; Crooke-Rosado, Jonathan L.; Cruz-García, José M.; Defendini-Ávila, Marianna; Delgado-Peraza, Francheska M.; Feliciano-Cancela, Alex J.; Gónzalez-Pérez, Valerie M.; Guiblet, Wilfried; Heredia-Negrón, Aldo; Hernández-Muñiz, Jennifer; Irizarry-González, Lourdes N.; Laboy-Corales, Ángel L.; Llaurador-Caraballo, Gabriela A.; Marín-Maldonado, Frances; Marrero-Llerena, Ulises; Martell-Martínez, Héctor A.; Martínez-Traverso, Idaliz M.; Medina-Ortega, Kiara N.; Méndez-Castellanos, Sonya G.; Menéndez-Serrano, Krizia C.; Morales-Caraballo, Carol I.; Ortiz-DeChoudens, Saryleine; Ortiz-Ortiz, Patricia; Pagán-Torres, Hendrick; Pérez-Afanador, Diana; Quintana-Torres, Enid M.; Ramírez-Aponte, Edwin G.; Riascos-Cuero, Carolina; Rivera-Llovet, Michelle S.; Rivera-Pagán, Ingrid T.; Rivera-Vicéns, Ramón E.; Robles-Juarbe, Fabiola; Rodríguez-Bonilla, Lorraine; Rodríguez-Echevarría, Brian O.; Rodríguez-García, Priscila M.; Rodríguez-Laboy, Abneris E.; Rodríguez-Santiago, Susana; Rojas-Vargas, Michael L.; Rubio-Marrero, Eva N.; Santiago-Colón, Albeliz; Santiago-Ortiz, Jorge L.; Santos-Ramos, Carlos E.; Serrano-González, Joseline; Tamayo-Figueroa, Alina M.; Tascón-Peñaranda, Edna P.; Torres-Castillo, José L.; Valentín-Feliciano, Nelson A.; Valentín-Feliciano, Yashira M.; Vargas-Barreto, Nadyan M.; Vélez-Vázquez, Miguel; Vilanova-Vélez, Luis R.; Zambrana-Echevarría, Cristina; MacKinnon, Christy; Chung, Hui-Min; Kay, Chris; Pinto, Anthony; Kopp, Olga R.; Burkhardt, Joshua; Harward, Chris; Allen, Robert; Bhat, Pavan; Chang, Jimmy Hsiang-Chun; Chen, York; Chesley, Christopher; Cohn, Dara; DuPuis, David; Fasano, Michael; Fazzio, Nicholas; Gavinski, Katherine; Gebreyesus, Heran; Giarla, Thomas; Gostelow, Marcus; Greenstein, Rachel; Gunasinghe, Hashini; Hanson, Casey; Hay, Amanda; He, Tao Jian; Homa, Katie; Howe, Ruth; Howenstein, Jeff; Huang, Henry; Khatri, Aaditya; Kim, Young Lu; Knowles, Olivia; Kong, Sarah; Krock, Rebecca; Kroll, Matt; Kuhn, Julia; Kwong, Matthew; Lee, Brandon; Lee, Ryan; Levine, Kevin; Li, Yedda; Liu, Bo; Liu, Lucy; Liu, Max; Lousararian, Adam; Ma, Jimmy; Mallya, Allyson; Manchee, Charlie; Marcus, Joseph; McDaniel, Stephen; Miller, Michelle L.; Molleston, Jerome M.; Diez, Cristina Montero; Ng, Patrick; Ngai, Natalie; Nguyen, Hien; Nylander, Andrew; Pollack, Jason; Rastogi, Suchita; Reddy, Himabindu; Regenold, Nathaniel; Sarezky, Jon; Schultz, Michael; Shim, Jien; Skorupa, Tara; Smith, Kenneth; Spencer, Sarah J.; Srikanth, Priya; Stancu, Gabriel; Stein, Andrew P.; Strother, Marshall; Sudmeier, Lisa; Sun, Mengyang; Sundaram, Varun; Tazudeen, Noor; Tseng, Alan; Tzeng, Albert; Venkat, Rohit; Venkataram, Sandeep; Waldman, Leah; Wang, Tracy; Yang, Hao; Yu, Jack Y.; Zheng, Yin; Preuss, Mary L.; Garcia, Angelica; Juergens, Matt; Morris, Robert W.; Nagengast, Alexis A.; Azarewicz, Julie; Carr, Thomas J.; Chichearo, Nicole; Colgan, Mike; Donegan, Megan; Gardner, Bob; Kolba, Nik; Krumm, Janice L.; Lytle, Stacey; MacMillian, Laurell; Miller, Mary; Montgomery, Andrew; Moretti, Alysha; Offenbacker, Brittney; Polen, Mike; Toth, John; Woytanowski, John; Kadlec, Lisa; Crawford, Justin; Spratt, Mary L.; Adams, Ashley L.; Barnard, Brianna K.; Cheramie, Martin N.; Eime, Anne M.; Golden, Kathryn L.; Hawkins, Allyson P.; Hill, Jessica E.; Kampmeier, Jessica A.; Kern, Cody D.; Magnuson, Emily E.; Miller, Ashley R.; Morrow, Cody M.; Peairs, Julia C.; Pickett, Gentry L.; Popelka, Sarah A.; Scott, Alexis J.; Teepe, Emily J.; TerMeer, Katie A.; Watchinski, Carmen A.; Watson, Lucas A.; Weber, Rachel E.; Woodard, Kate A.; Barnard, Daron C.; Appiah, Isaac; Giddens, Michelle M.; McNeil, Gerard P.; Adebayo, Adeola; Bagaeva, Kate; Chinwong, Justina; Dol, Chrystel; George, Eunice; Haltaufderhyde, Kirk; Haye, Joanna; Kaur, Manpreet; Semon, Max; Serjanov, Dmitri; Toorie, Anika; Wilson, Christopher; Riddle, Nicole C.; Buhler, Jeremy; Mardis, Elaine R.

    2015-01-01

    The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu. PMID:25740935

  3. Multiple controls affect arsenite oxidase gene expression in Herminiimonas arsenicoxydans

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    Coppée Jean-Yves

    2010-02-01

    Full Text Available Abstract Background Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. Herminiimonas arsenicoxydans has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III to As(V as a detoxification mechanism. Results In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of H. arsenicoxydans to As(III. To get insight into the molecular mechanisms of this enzyme activity, a Tn5 transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted aoxR and aoxS genes, showing that the aox operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in rpoN coding for the alternative N sigma factor (σ54 of RNA polymerase and in dnaJ coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the rpoN and dnaJ gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the aoxAB operon was determined using rapid amplification of cDNA ends (RACE and a putative -12/-24 σ54-dependent promoter motif was identified upstream of aoxAB coding sequences. Conclusion These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in H. arsenicoxydans. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III in this microorganism.

  4. Signature-tagged mutagenesis screening revealed a novel smooth-to-rough transition determinant of Salmonella enterica serovar Enteritidis.

    Science.gov (United States)

    Jiao, Yang; Guo, Rongxian; Tang, Peipei; Kang, Xilong; Yin, Junlei; Wu, Kaiyue; Geng, Shizhong; Li, Qiuchun; Sun, Jun; Xu, Xiulong; Zhou, Xiaohui; Gan, Junji; Jiao, Xinan; Liu, Xiufan; Pan, Zhiming

    2017-03-03

    Salmonella enterica serovar Enteritidis (S. Enteritidis) has emerged as one of the most important food-borne pathogens for humans. Lipopolysaccharide (LPS), as a component of the outer membrane, is responsible for the virulence and smooth-to-rough transition in S. Enteritidis. In this study, we screened S. Enteritidis signature-tagged transposon mutant library using monoclonal antibody against somatic O 9 antigen (O 9 MAb) and O 9 factor rabbit antiserum to identify novel genes that are involved in smooth-to-rough transition. A total of 480 mutants were screened and one mutant with transposon insertion in rfbG gene had smooth-to-rough transition phenotype. In order to verify the role of rfbG gene, an rfbG insertion or deletion mutant was constructed using λ-Red recombination system. Phenotypic and biological analysis revealed that rfbG insertion or deletion mutants were similar to the wild-type strain in growth rate and biochemical properties, but the swimming motility was reduced. SE Slide Agglutination test and ELISA test showed that rfbG mutants do not stimulate animals to produce agglutinating antibody. In addition, the half-lethal dose (LD 50 ) of the rfbG deletion mutant strain was 10 6.6 -fold higher than that of the parent strain in a mouse model when injected intraperitoneally. These data indicate that the rfbG gene is involved in smooth-to-rough transition, swimming motility and virulence of S. Enteritidis. Furthermore, somatic O-antigen antibody-based approach to screen signature-tagged transposon mutants is feasible to clarify LPS biosynthesis and to find suitable markers in DIVA-vaccine research.

  5. In vivo mapping of the functional regions of the DEAD-box helicase Vasa

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    Mehrnoush Dehghani

    2015-03-01

    Full Text Available The maternally expressed Drosophila melanogaster DEAD-box helicase Vasa (Vas is necessary for many cellular and developmental processes, including specification of primordial germ cells (pole cells, posterior patterning of the embryo, piRNA-mediated repression of transposon-encoded mRNAs, translational activation of gurken (grk mRNA, and completion of oogenesis itself. Vas protein accumulates in the perinuclear nuage in nurse cells soon after their specification, and then at stage 10 Vas translocates to the posterior pole plasm of the oocyte. We produced a series of transgenic constructs encoding eGFP-Vas proteins carrying mutations affecting different regions of the protein, and analyzed in vivo which Vas functions each could support. We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification. One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases. Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression. We conclude from these experiments that Vas, a multifunctional protein, uses different domains and different molecular associations to carry out its various cellular and developmental roles.

  6. ManA is regulated by RssAB signaling and promotes motility in Serratia marcescens.

    Science.gov (United States)

    Soo, Po-Chi; Horng, Yu-Tze; Chang, Yung-Lin; Tsai, Wei-Wen; Jeng, Wen-Yih; Lu, Chia-Chen; Lai, Hsin-Chih

    2014-01-01

    Serratia marcescens swarms on 0.8% LB agar at 30 °C but not at 37 °C. To understand the molecular mechanism regulating Serratia swarming, transposon mutagenesis was performed to screen for mutants that swarmed at 37 °C. In one mutant, S. marcescens WW100, the transposon was inserted in the upstream region of manA, which encodes mannose-6-phosphate isomerase, a type I phosphomannose isomerase. The transcriptional and translational levels of manA were higher in S. marcescens WW100 than in the wild-type strain. S. marcescens WW100 produced more serrawettin W1 (biosurfactant) than the wild-type, as detected by thin-layer chromatography, to promote surface motility by reducing surface tension. Serratia swarming was previously shown to be negatively regulated by the RssA-RssB two-component system. An electrophoretic mobility shift assay (EMSA) indicated that phosphorylated RssB (the response regulator) binds upstream of the transposon insertion site and manA in S. marcescens WW100. Analysis by real-time RT-PCR (qRT-PCR) revealed that, compared to the wild-type level, manA mRNA was increased in the rssA deletion mutant. The results indicated that RssA-RssB signaling directly represses the expression of manA and that overexpression of manA increases the production of serrawettin for Serratia swarming at 37 °C. Copyright © 2013 Institut Pasteur. All rights reserved.

  7. Expression of the gene cluster associated with the Escherichia coli pilus adhesin K99.

    Science.gov (United States)

    Lee, J H; Isaacson, R E

    1995-10-01

    The biogenesis of the pilus adhesin K99 is dependent on the expression of eight contiguous genes, fanA to fanH. Transposon mutants were prepared by using TnlacZ and TnphoA, and selected transposon mutants were used to measure expression of each K99 gene. Expression of the K99 genes is likely controlled at the transcription level, since in general, there were no differences between the results obtained with the two transposons. fanC was the most highly expressed, and fanD was expressed at very low levels. The expression of TnlacZ fusions in fanA and fanB fusions was high. Deletion of fanA, fanB, and part of fanC abolished the expression of fanD but had no effect on the distal genes fanE to fanH. To locate the DNA regions required for expression of fanE to fanH, deletion mutations were prepared and the effects on expression of fanE to fanH were determined. The deletion of a segment between fanD and fanE abolished fanE and fanF expression but did not affect fanG and fanH. The deletion of a portion of fanF (approximately 1 kb proximal to fanG) abolished the expression of fanG and fanH. These results indicate the presence of regulatory elements proximal to fanE and to fanG. Putative promoters were identified in these regions by DNA homology and by primer extension. A stem-loop structure that may act as a transcriptional attenuator of fanF was also found at the beginning of fanF. These data confirm our previous model of K99 transcriptional organization.

  8. Lipooligosaccharide structure is an important determinant in the resistance of Neisseria gonorrhoeae to antimicrobial agents of innate host defense

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    Jacqueline T Balthazar

    2011-02-01

    Full Text Available The strict human pathogen Neisseria gonorrhoeae has caused the sexually transmitted infection termed gonorrhea for thousands of years. Over the millennia, the gonococcus has likely evolved mechanisms to evade host defense systems that operate on the genital mucosal surfaces in both males and females. Past research has shown that the presence or modification of certain cell envelope structures can significantly impact levels of gonococcal susceptibility to host-derived antimicrobial compounds that bathe genital mucosal surfaces and participate in innate host defense against invading pathogens. In order to facilitate the identification of gonococcal genes that are important in determining levels of bacterial susceptibility to mediators of innate host defense, we used the Himar I mariner in vitro mutagenesis system to construct a transposon insertion library in strain F62. As proof of principle that this strategy would be suitable for this purpose, we screened the library for mutants expressing decreased susceptibility to the bacteriolytic action of normal human serum (NHS. We found that a transposon insertion in the lgtD gene, which encodes an N-acetylgalactosamine transferase involved in the extension of the α-chain of lipooligosaccharide (LOS, could confer decreased susceptibility of strain F62 to complement-mediated killing by NHS. By complementation and chemical analyses, we demonstrated both linkage of the transposon insertion to the NHS-resistance phenotype and chemical changes in LOS structure that resulted from loss of LgtD production. Further truncation of the LOS α-chain or loss of phosphoethanolamine (PEA from the lipid A region of LOS also impacted levels of NHS-resistance. PEA decoration of lipid A also increased gonococcal resistance to the model cationic antimicrobial polymyxin B. Taken together, we conclude that the Himar I mariner in vitro mutagenesis procedure can facilitate studies on structures involved in gonococcal

  9. Identification of genes required for secretion of the Francisella oxidative burst-inhibiting acid phosphatase AcpA

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    John S Gunn

    2016-04-01

    Full Text Available Francisella tularensis is a Tier 1 bioterror threat and the intracellular pathogen responsible for tularemia in humans and animals. Upon entry into the host, Francisella uses multiple mechanisms to evade killing. Our previous studies have shown that after entering its primary cellular host, the macrophage, Francisella immediately suppresses the oxidative burst by secreting a series of acid phosphatases including AcpA-B-C and HapA, thereby evading the innate immune response of the macrophage and enhancing survival and further infection. However, the mechanism of acid phosphatase secretion by Francisella is still unknown. In this study, we screened for genes required for AcpA secretion in Francisella. We initially demonstrated that the known secretion systems, the putative Francisella-pathogenicity island (FPI-encoded Type VI secretion system and the Type IV pili, do not secrete AcpA. Using random transposon mutagenesis in conjunction with ELISA, Western blotting and acid phosphatase enzymatic assays, a transposon library of 5450 mutants was screened for strains with a minimum 1.5-fold decrease in secreted (culture supernatant AcpA, but no defect in cytosolic AcpA. Three mutants with decreased supernatant AcpA were identified. The transposon insertion sites of these mutants were revealed by direct genomic sequencing or inverse-PCR and sequencing. One of these mutants has a severe defect in AcpA secretion (at least 85% decrease and is a predicted hypothetical inner membrane protein. Interestingly, this mutant also affected the secretion of the FPI-encoded protein, VgrG. Thus, this screen identified novel protein secretion factors involved in the subversion of host defenses.

  10. Fitness of isogenic colony morphology variants of Pseudomonas aeruginosa in murine airway infection.

    Directory of Open Access Journals (Sweden)

    Elza Rakhimova

    Full Text Available Chronic lung infections with Pseudomonas aeruginosa are associated with the diversification of the persisting clone into niche specialists and morphotypes, a phenomenon called 'dissociative behaviour'. To explore the potential of P. aeruginosa to change its morphotype by single step loss-of-function mutagenesis, a signature-tagged mini-Tn5 plasposon library of the cystic fibrosis airway isolate TBCF10839 was screened for colony morphology variants under nine different conditions in vitro. Transposon insertion into 1% of the genome changed colony morphology into eight discernable morphotypes. Half of the 55 targets encode features of primary or secondary metabolism whereby quinolone production was frequently affected. In the other half the transposon had inserted into genes of the functional categories transport, regulation or motility/chemotaxis. To mimic dissociative behaviour of isogenic strains in lungs, pools of 25 colony morphology variants were tested for competitive fitness in an acute murine airway infection model. Six of the 55 mutants either grew better or worse in vivo than in vitro, respectively. Metabolic proficiency of the colony morphology variant was a key determinant for survival in murine airways. The most common morphotype of self-destructive autolysis did unexpectedly not impair fitness. Transposon insertions into homologous genes of strain PAO1 did not reproduce the TBCF10839 mutant morphotypes for 16 of 19 examined loci pointing to an important role of the genetic background on colony morphology. Depending on the chosen P. aeruginosa strain, functional genome scans will explore other areas of the evolutionary landscape. Based on our discordant findings of mutant phenotypes in P. aeruginosa strains PAO1, PA14 and TBCF10839, we conclude that the current focus on few reference strains may miss modes of niche adaptation and dissociative behaviour that are relevant for the microevolution of complex traits in the wild.

  11. Genetic Control of Plant Root Colonization by the Biocontrol agent, Pseudomonas fluorescens

    Energy Technology Data Exchange (ETDEWEB)

    Cole, Benjamin J.; Fletcher, Meghan; Waters, Jordan; Wetmore, Kelly; Blow, Matthew J.; Deutschbauer, Adam M.; Dangl, Jeffry L.; Visel, Axel

    2015-03-19

    Plant growth promoting rhizobacteria (PGPR) are a critical component of plant root ecosystems. PGPR promote plant growth by solubilizing inaccessible minerals, suppressing pathogenic microorganisms in the soil, and directly stimulating growth through hormone synthesis. Pseudomonas fluorescens is a well-established PGPR isolated from wheat roots that can also colonize the root system of the model plant, Arabidopsis thaliana. We have created barcoded transposon insertion mutant libraries suitable for genome-wide transposon-mediated mutagenesis followed by sequencing (TnSeq). These libraries consist of over 105 independent insertions, collectively providing loss-of-function mutants for nearly all genes in the P.fluorescens genome. Each insertion mutant can be unambiguously identified by a randomized 20 nucleotide sequence (barcode) engineered into the transposon sequence. We used these libraries in a gnotobiotic assay to examine the colonization ability of P.fluorescens on A.thaliana roots. Taking advantage of the ability to distinguish individual colonization events using barcode sequences, we assessed the timing and microbial concentration dependence of colonization of the rhizoplane niche. These data provide direct insight into the dynamics of plant root colonization in an in vivo system and define baseline parameters for the systematic identification of the bacterial genes and molecular pathways using TnSeq assays. Having determined parameters that facilitate potential colonization of roots by thousands of independent insertion mutants in a single assay, we are currently establishing a genome-wide functional map of genes required for root colonization in P.fluorescens. Importantly, the approach developed and optimized here for P.fluorescens>A.thaliana colonization will be applicable to a wide range of plant-microbe interactions, including biofuel feedstock plants and microbes known or hypothesized to impact on biofuel-relevant traits including biomass productivity

  12. Drosophila muller f elements maintain a distinct set of genomic properties over 40 million years of evolution.

    Science.gov (United States)

    Leung, Wilson; Shaffer, Christopher D; Reed, Laura K; Smith, Sheryl T; Barshop, William; Dirkes, William; Dothager, Matthew; Lee, Paul; Wong, Jeannette; Xiong, David; Yuan, Han; Bedard, James E J; Machone, Joshua F; Patterson, Seantay D; Price, Amber L; Turner, Bryce A; Robic, Srebrenka; Luippold, Erin K; McCartha, Shannon R; Walji, Tezin A; Walker, Chelsea A; Saville, Kenneth; Abrams, Marita K; Armstrong, Andrew R; Armstrong, William; Bailey, Robert J; Barberi, Chelsea R; Beck, Lauren R; Blaker, Amanda L; Blunden, Christopher E; Brand, Jordan P; Brock, Ethan J; Brooks, Dana W; Brown, Marie; Butzler, Sarah C; Clark, Eric M; Clark, Nicole B; Collins, Ashley A; Cotteleer, Rebecca J; Cullimore, Peterson R; Dawson, Seth G; Docking, Carter T; Dorsett, Sasha L; Dougherty, Grace A; Downey, Kaitlyn A; Drake, Andrew P; Earl, Erica K; Floyd, Trevor G; Forsyth, Joshua D; Foust, Jonathan D; Franchi, Spencer L; Geary, James F; Hanson, Cynthia K; Harding, Taylor S; Harris, Cameron B; Heckman, Jonathan M; Holderness, Heather L; Howey, Nicole A; Jacobs, Dontae A; Jewell, Elizabeth S; Kaisler, Maria; Karaska, Elizabeth A; Kehoe, James L; Koaches, Hannah C; Koehler, Jessica; Koenig, Dana; Kujawski, Alexander J; Kus, Jordan E; Lammers, Jennifer A; Leads, Rachel R; Leatherman, Emily C; Lippert, Rachel N; Messenger, Gregory S; Morrow, Adam T; Newcomb, Victoria; Plasman, Haley J; Potocny, Stephanie J; Powers, Michelle K; Reem, Rachel M; Rennhack, Jonathan P; Reynolds, Katherine R; Reynolds, Lyndsey A; Rhee, Dong K; Rivard, Allyson B; Ronk, Adam J; Rooney, Meghan B; Rubin, Lainey S; Salbert, Luke R; Saluja, Rasleen K; Schauder, Taylor; Schneiter, Allison R; Schulz, Robert W; Smith, Karl E; Spencer, Sarah; Swanson, Bryant R; Tache, Melissa A; Tewilliager, Ashley A; Tilot, Amanda K; VanEck, Eve; Villerot, Matthew M; Vylonis, Megan B; Watson, David T; Wurzler, Juliana A; Wysocki, Lauren M; Yalamanchili, Monica; Zaborowicz, Matthew A; Emerson, Julia A; Ortiz, Carlos; Deuschle, Frederic J; DiLorenzo, Lauren A; Goeller, Katie L; Macchi, Christopher R; Muller, Sarah E; Pasierb, Brittany D; Sable, Joseph E; Tucci, Jessica M; Tynon, Marykathryn; Dunbar, David A; Beken, Levent H; Conturso, Alaina C; Danner, Benjamin L; DeMichele, Gabriella A; Gonzales, Justin A; Hammond, Maureen S; Kelley, Colleen V; Kelly, Elisabeth A; Kulich, Danielle; Mageeney, Catherine M; McCabe, Nikie L; Newman, Alyssa M; Spaeder, Lindsay A; Tumminello, Richard A; Revie, Dennis; Benson, Jonathon M; Cristostomo, Michael C; DaSilva, Paolo A; Harker, Katherine S; Jarrell, Jenifer N; Jimenez, Luis A; Katz, Brandon M; Kennedy, William R; Kolibas, Kimberly S; LeBlanc, Mark T; Nguyen, Trung T; Nicolas, Daniel S; Patao, Melissa D; Patao, Shane M; Rupley, Bryan J; Sessions, Bridget J; Weaver, Jennifer A; Goodman, Anya L; Alvendia, Erica L; Baldassari, Shana M; Brown, Ashley S; Chase, Ian O; Chen, Maida; Chiang, Scott; Cromwell, Avery B; Custer, Ashley F; DiTommaso, Tia M; El-Adaimi, Jad; Goscinski, Nora C; Grove, Ryan A; Gutierrez, Nestor; Harnoto, Raechel S; Hedeen, Heather; Hong, Emily L; Hopkins, Barbara L; Huerta, Vilma F; Khoshabian, Colin; LaForge, Kristin M; Lee, Cassidy T; Lewis, Benjamin M; Lydon, Anniken M; Maniaci, Brian J; Mitchell, Ryan D; Morlock, Elaine V; Morris, William M; Naik, Priyanka; Olson, Nicole C; Osterloh, Jeannette M; Perez, Marcos A; Presley, Jonathan D; Randazzo, Matt J; Regan, Melanie K; Rossi, Franca G; Smith, Melanie A; Soliterman, Eugenia A; Sparks, Ciani J; Tran, Danny L; Wan, Tiffany; Welker, Anne A; Wong, Jeremy N; Sreenivasan, Aparna; Youngblom, Jim; Adams, Andrew; Alldredge, Justin; Bryant, Ashley; Carranza, David; Cifelli, Alyssa; Coulson, Kevin; Debow, Calise; Delacruz, Noelle; Emerson, Charlene; Farrar, Cassandra; Foret, Don; Garibay, Edgar; Gooch, John; Heslop, Michelle; Kaur, Sukhjit; Khan, Ambreen; Kim, Van; Lamb, Travis; Lindbeck, Peter; Lucas, Gabi; Macias, Elizabeth; Martiniuc, Daniela; Mayorga, Lissett; Medina, Joseph; Membreno, Nelson; Messiah, Shady; Neufeld, Lacey; Nguyen, San Francisco; Nichols, Zachary; Odisho, George; Peterson, Daymon; Rodela, Laura; Rodriguez, Priscilla; Rodriguez, Vanessa; Ruiz, Jorge; Sherrill, Will; Silva, Valeria; Sparks, Jeri; Statton, Geeta; Townsend, Ashley; Valdez, Isabel; Waters, Mary; Westphal, Kyle; Winkler, Stacey; Zumkehr, Joannee; DeJong, Randall J; Hoogewerf, Arlene J; Ackerman, Cheri M; Armistead, Isaac O; Baatenburg, Lara; Borr, Matthew J; Brouwer, Lindsay K; Burkhart, Brandon J; Bushhouse, Kelsey T; Cesko, Lejla; Choi, Tiffany Y Y; Cohen, Heather; Damsteegt, Amanda M; Darusz, Jess M; Dauphin, Cory M; Davis, Yelena P; Diekema, Emily J; Drewry, Melissa; Eisen, Michelle E M; Faber, Hayley M; Faber, Katherine J; Feenstra, Elizabeth; Felzer-Kim, Isabella T; Hammond, Brandy L; Hendriksma, Jesse; Herrold, Milton R; Hilbrands, Julia A; Howell, Emily J; Jelgerhuis, Sarah A; Jelsema, Timothy R; Johnson, Benjamin K; Jones, Kelly K; Kim, Anna; Kooienga, Ross D; Menyes, Erika E; Nollet, Eric A; Plescher, Brittany E; Rios, Lindsay; Rose, Jenny L; Schepers, Allison J; Scott, Geoff; Smith, Joshua R; Sterling, Allison M; Tenney, Jenna C; Uitvlugt, Chris; VanDyken, Rachel E; VanderVennen, Marielle; Vue, Samantha; Kokan, Nighat P; Agbley, Kwabea; Boham, Sampson K; Broomfield, Daniel; Chapman, Kayla; Dobbe, Ali; Dobbe, Ian; Harrington, William; Ibrahem, Marwan; Kennedy, Andre; Koplinsky, Chad A; Kubricky, Cassandra; Ladzekpo, Danielle; Pattison, Claire; Ramirez, Roman E; Wande, Lucia; Woehlke, Sarah; Wawersik, Matthew; Kiernan, Elizabeth; Thompson, Jeffrey S; Banker, Roxanne; Bartling, Justina R; Bhatiya, Chinmoy I; Boudoures, Anna L; Christiansen, Lena; Fosselman, Daniel S; French, Kristin M; Gill, Ishwar S; Havill, Jessen T; Johnson, Jaelyn L; Keny, Lauren J; Kerber, John M; Klett, Bethany M; Kufel, Christina N; May, Francis J; Mecoli, Jonathan P; Merry, Callie R; Meyer, Lauren R; Miller, Emily G; Mullen, Gregory J; Palozola, Katherine C; Pfeil, Jacob J; Thomas, Jessica G; Verbofsky, Evan M; Spana, Eric P; Agarwalla, Anant; Chapman, Julia; Chlebina, Ben; Chong, Insun; Falk, I N; Fitzgibbons, John D; Friedman, Harrison; Ighile, Osagie; Kim, Andrew J; Knouse, Kristin A; Kung, Faith; Mammo, Danny; Ng, Chun Leung; Nikam, Vinayak S; Norton, Diana; Pham, Philip; Polk, Jessica W; Prasad, Shreya; Rankin, Helen; Ratliff, Camille D; Scala, Victoria; Schwartz, Nicholas U; Shuen, Jessica A; Xu, Amy; Xu, Thomas Q; Zhang, Yi; Rosenwald, Anne G; Burg, Martin G; Adams, Stephanie J; Baker, Morgan; Botsford, Bobbi; Brinkley, Briana; Brown, Carter; Emiah, Shadie; Enoch, Erica; Gier, Chad; Greenwell, Alyson; Hoogenboom, Lindsay; Matthews, Jordan E; McDonald, Mitchell; Mercer, Amanda; Monsma, Nicholaus; Ostby, Kristine; Ramic, Alen; Shallman, Devon; Simon, Matthew; Spencer, Eric; Tomkins, Trisha; Wendland, Pete; Wylie, Anna; Wolyniak, Michael J; Robertson, Gregory M; Smith, Samuel I; DiAngelo, Justin R; Sassu, Eric D; Bhalla, Satish C; Sharif, Karim A; Choeying, Tenzin; Macias, Jason S; Sanusi, Fareed; Torchon, Karvyn; Bednarski, April E; Alvarez, Consuelo J; Davis, Kristen C; Dunham, Carrie A; Grantham, Alaina J; Hare, Amber N; Schottler, Jennifer; Scott, Zackary W; Kuleck, Gary A; Yu, Nicole S; Kaehler, Marian M; Jipp, Jacob; Overvoorde, Paul J; Shoop, Elizabeth; Cyrankowski, Olivia; Hoover, Betsy; Kusner, Matt; Lin, Devry; Martinov, Tijana; Misch, Jonathan; Salzman, Garrett; Schiedermayer, Holly; Snavely, Michael; Zarrasola, Stephanie; Parrish, Susan; Baker, Atlee; Beckett, Alissa; Belella, Carissa; Bryant, Julie; Conrad, Turner; Fearnow, Adam; Gomez, Carolina; Herbstsomer, Robert A; Hirsch, Sarah; Johnson, Christen; Jones, Melissa; Kabaso, Rita; Lemmon, Eric; Vieira, Carolina Marques Dos Santos; McFarland, Darryl; McLaughlin, Christopher; Morgan, Abbie; Musokotwane, Sepo; Neutzling, William; Nietmann, Jana; Paluskievicz, Christina; Penn, Jessica; Peoples, Emily; Pozmanter, Caitlin; Reed, Emily; Rigby, Nichole; Schmidt, Lasse; Shelton, Micah; Shuford, Rebecca; Tirasawasdichai, Tiara; Undem, Blair; Urick, Damian; Vondy, Kayla; Yarrington, Bryan; Eckdahl, Todd T; Poet, Jeffrey L; Allen, Alica B; Anderson, John E; Barnett, Jason M; Baumgardner, Jordan S; Brown, Adam D; Carney, Jordan E; Chavez, Ramiro A; Christgen, Shelbi L; Christie, Jordan S; Clary, Andrea N; Conn, Michel A; Cooper, Kristen M; Crowley, Matt J; Crowley, Samuel T; Doty, Jennifer S; Dow, Brian A; Edwards, Curtis R; Elder, Darcie D; Fanning, John P; Janssen, Bridget M; Lambright, Anthony K; Lane, Curtiss E; Limle, Austin B; Mazur, Tammy; McCracken, Marly R; McDonough, Alexa M; Melton, Amy D; Minnick, Phillip J; Musick, Adam E; Newhart, William H; Noynaert, Joseph W; Ogden, Bradley J; Sandusky, Michael W; Schmuecker, Samantha M; Shipman, Anna L; Smith, Anna L; Thomsen, Kristen M; Unzicker, Matthew R; Vernon, William B; Winn, Wesley W; Woyski, Dustin S; Zhu, Xiao; Du, Chunguang; Ament, Caitlin; Aso, Soham; Bisogno, Laura Simone; Caronna, Jason; Fefelova, Nadezhda; Lopez, Lenin; Malkowitz, Lorraine; Marra, Jonathan; Menillo, Daniella; Obiorah, Ifeanyi; Onsarigo, Eric Nyabeta; Primus, Shekerah; Soos, Mahdi; Tare, Archana; Zidan, Ameer; Jones, Christopher J; Aronhalt, Todd; Bellush, James M; Burke, Christa; DeFazio, Steve; Does, Benjamin R; Johnson, Todd D; Keysock, Nicholas; Knudsen, Nelson H; Messler, James; Myirski, Kevin; Rekai, Jade Lea; Rempe, Ryan Michael; Salgado, Michael S; Stagaard, Erica; Starcher, Justin R; Waggoner, Andrew W; Yemelyanova, Anastasia K; Hark, Amy T; Bertolet, Anne; Kuschner, Cyrus E; Parry, Kesley; Quach, Michael; Shantzer, Lindsey; Shaw, Mary E; Smith, Mary A; Glenn, Omolara; Mason, Portia; Williams, Charlotte; Key, S Catherine Silver; Henry, Tyneshia C P; Johnson, Ashlee G; White, Jackie X; Haberman, Adam; Asinof, Sam; Drumm, Kelly; Freeburg, Trip; Safa, Nadia; Schultz, Darrin; Shevin, Yakov; Svoronos, Petros; Vuong, Tam; Wellinghoff, Jules; Hoopes, Laura L M; Chau, Kim M; Ward, Alyssa; Regisford, E Gloria C; Augustine, LaJerald; Davis-Reyes, Brionna; Echendu, Vivienne; Hales, Jasmine; Ibarra, Sharon; Johnson, Lauriaun; Ovu, Steven; Braverman, John M; Bahr, Thomas J; Caesar, Nicole M; Campana, Christopher; Cassidy, Daniel W; Cognetti, Peter A; English, Johnathan D; Fadus, Matthew C; Fick, Cameron N; Freda, Philip J; Hennessy, Bryan M; Hockenberger, Kelsey; Jones, Jennifer K; King, Jessica E; Knob, Christopher R; Kraftmann, Karen J; Li, Linghui; Lupey, Lena N; Minniti, Carl J; Minton, Thomas F; Moran, Joseph V; Mudumbi, Krishna; Nordman, Elizabeth C; Puetz, William J; Robinson, Lauren M; Rose, Thomas J; Sweeney, Edward P; Timko, Ashley S; Paetkau, Don W; Eisler, Heather L; Aldrup, Megan E; Bodenberg, Jessica M; Cole, Mara G; Deranek, Kelly M; DeShetler, Megan; Dowd, Rose M; Eckardt, Alexandra K; Ehret, Sharon C; Fese, Jessica; Garrett, Amanda D; Kammrath, Anna; Kappes, Michelle L; Light, Morgan R; Meier, Anne C; O'Rouke, Allison; Perella, Mallory; Ramsey, Kimberley; Ramthun, Jennifer R; Reilly, Mary T; Robinett, Deirdre; Rossi, Nadine L; Schueler, Mary Grace; Shoemaker, Emma; Starkey, Kristin M; Vetor, Ashley; Vrable, Abby; Chandrasekaran, Vidya; Beck, Christopher; Hatfield, Kristen R; Herrick, Douglas A; Khoury, Christopher B; Lea, Charlotte; Louie, Christopher A; Lowell, Shannon M; Reynolds, Thomas J; Schibler, Jeanine; Scoma, Alexandra H; Smith-Gee, Maxwell T; Tuberty, Sarah; Smith, Christopher D; Lopilato, Jane E; Hauke, Jeanette; Roecklein-Canfield, Jennifer A; Corrielus, Maureen; Gilman, Hannah; Intriago, Stephanie; Maffa, Amanda; Rauf, Sabya A; Thistle, Katrina; Trieu, Melissa; Winters, Jenifer; Yang, Bib; Hauser, Charles R; Abusheikh, Tariq; Ashrawi, Yara; Benitez, Pedro; Boudreaux, Lauren R; Bourland, Megan; Chavez, Miranda; Cruz, Samantha; Elliott, GiNell; Farek, Jesse R; Flohr, Sarah; Flores, Amanda H; Friedrichs, Chelsey; Fusco, Zach; Goodwin, Zane; Helmreich, Eric; Kiley, John; Knepper, John Mark; Langner, Christine; Martinez, Megan; Mendoza, Carlos; Naik, Monal; Ochoa, Andrea; Ragland, Nicolas; Raimey, England; Rathore, Sunil; Reza, Evangelina; Sadovsky, Griffin; Seydoux, Marie-Isabelle B; Smith, Jonathan E; Unruh, Anna K; Velasquez, Vicente; Wolski, Matthew W; Gosser, Yuying; Govind, Shubha; Clarke-Medley, Nicole; Guadron, Leslie; Lau, Dawn; Lu, Alvin; Mazzeo, Cheryl; Meghdari, Mariam; Ng, Simon; Pamnani, Brad; Plante, Olivia; Shum, Yuki Kwan Wa; Song, Roy; Johnson, Diana E; Abdelnabi, Mai; Archambault, Alexi; Chamma, Norma; Gaur, Shailly; Hammett, Deborah; Kandahari, Adrese; Khayrullina, Guzal; Kumar, Sonali; Lawrence, Samantha; Madden, Nigel; Mandelbaum, Max; Milnthorp, Heather; Mohini, Shiv; Patel, Roshni; Peacock, Sarah J; Perling, Emily; Quintana, Amber; Rahimi, Michael; Ramirez, Kristen; Singhal, Rishi; Weeks, Corinne; Wong, Tiffany; Gillis, Aubree T; Moore, Zachary D; Savell, Christopher D; Watson, Reece; Mel, Stephanie F; Anilkumar, Arjun A; Bilinski, Paul; Castillo, Rostislav; Closser, Michael; Cruz, Nathalia M; Dai, Tiffany; Garbagnati, Giancarlo F; Horton, Lanor S; Kim, Dongyeon; Lau, Joyce H; Liu, James Z; Mach, Sandy D; Phan, Thu A; Ren, Yi; Stapleton, Kenneth E; Strelitz, Jean M; Sunjed, Ray; Stamm, Joyce; Anderson, Morgan C; Bonifield, Bethany Grace; Coomes, Daniel; Dillman, Adam; Durchholz, Elaine J; Fafara-Thompson, Antoinette E; Gross, Meleah J; Gygi, Amber M; Jackson, Lesley E; Johnson, Amy; Kocsisova, Zuzana; Manghelli, Joshua L; McNeil, Kylie; Murillo, Michael; Naylor, Kierstin L; Neely, Jessica; Ogawa, Emmy E; Rich, Ashley; Rogers, Anna; Spencer, J Devin; Stemler, Kristina M; Throm, Allison A; Van Camp, Matt; Weihbrecht, Katie; Wiles, T Aaron; Williams, Mallory A; Williams, Matthew; Zoll, Kyle; Bailey, Cheryl; Zhou, Leming; Balthaser, Darla M; Bashiri, Azita; Bower, Mindy E; Florian, Kayla A; Ghavam, Nazanin; Greiner-Sosanko, Elizabeth S; Karim, Helmet; Mullen, Victor W; Pelchen, Carly E; Yenerall, Paul M; Zhang, Jiayu; Rubin, Michael R; Arias-Mejias, Suzette M; Bermudez-Capo, Armando G; Bernal-Vega, Gabriela V; Colon-Vazquez, Mariela; Flores-Vazquez, Arelys; Gines-Rosario, Mariela; Llavona-Cartagena, Ivan G; Martinez-Rodriguez, Javier O; Ortiz-Fuentes, Lionel; Perez-Colomba, Eliezer O; Perez-Otero, Joseph; Rivera, Elisandra; Rodriguez-Giron, Luke J; Santiago-Sanabria, Arnaldo J; Senquiz-Gonzalez, Andrea M; delValle, Frank R Soto; Vargas-Franco, Dorianmarie; Velázquez-Soto, Karla I; Zambrana-Burgos, Joan D; Martinez-Cruzado, Juan Carlos; Asencio-Zayas, Lillyann; Babilonia-Figueroa, Kevin; Beauchamp-Pérez, Francis D; Belén-Rodríguez, Juliana; Bracero-Quiñones, Luciann; Burgos-Bula, Andrea P; Collado-Méndez, Xavier A; Colón-Cruz, Luis R; Correa-Muller, Ana I; Crooke-Rosado, Jonathan L; Cruz-García, José M; Defendini-Ávila, Marianna; Delgado-Peraza, Francheska M; Feliciano-Cancela, Alex J; Gónzalez-Pérez, Valerie M; Guiblet, Wilfried; Heredia-Negrón, Aldo; Hernández-Muñiz, Jennifer; Irizarry-González, Lourdes N; Laboy-Corales, Ángel L; Llaurador-Caraballo, Gabriela A; Marín-Maldonado, Frances; Marrero-Llerena, Ulises; Martell-Martínez, Héctor A; Martínez-Traverso, Idaliz M; Medina-Ortega, Kiara N; Méndez-Castellanos, Sonya G; Menéndez-Serrano, Krizia C; Morales-Caraballo, Carol I; Ortiz-DeChoudens, Saryleine; Ortiz-Ortiz, Patricia; Pagán-Torres, Hendrick; Pérez-Afanador, Diana; Quintana-Torres, Enid M; Ramírez-Aponte, Edwin G; Riascos-Cuero, Carolina; Rivera-Llovet, Michelle S; Rivera-Pagán, Ingrid T; Rivera-Vicéns, Ramón E; Robles-Juarbe, Fabiola; Rodríguez-Bonilla, Lorraine; Rodríguez-Echevarría, Brian O; Rodríguez-García, Priscila M; Rodríguez-Laboy, Abneris E; Rodríguez-Santiago, Susana; Rojas-Vargas, Michael L; Rubio-Marrero, Eva N; Santiago-Colón, Albeliz; Santiago-Ortiz, Jorge L; Santos-Ramos, Carlos E; Serrano-González, Joseline; Tamayo-Figueroa, Alina M; Tascón-Peñaranda, Edna P; Torres-Castillo, José L; Valentín-Feliciano, Nelson A; Valentín-Feliciano, Yashira M; Vargas-Barreto, Nadyan M; Vélez-Vázquez, Miguel; Vilanova-Vélez, Luis R; Zambrana-Echevarría, Cristina; MacKinnon, Christy; Chung, Hui-Min; Kay, Chris; Pinto, Anthony; Kopp, Olga R; Burkhardt, Joshua; Harward, Chris; Allen, Robert; Bhat, Pavan; Chang, Jimmy Hsiang-Chun; Chen, York; Chesley, Christopher; Cohn, Dara; DuPuis, David; Fasano, Michael; Fazzio, Nicholas; Gavinski, Katherine; Gebreyesus, Heran; Giarla, Thomas; Gostelow, Marcus; Greenstein, Rachel; Gunasinghe, Hashini; Hanson, Casey; Hay, Amanda; He, Tao Jian; Homa, Katie; Howe, Ruth; Howenstein, Jeff; Huang, Henry; Khatri, Aaditya; Kim, Young Lu; Knowles, Olivia; Kong, Sarah; Krock, Rebecca; Kroll, Matt; Kuhn, Julia; Kwong, Matthew; Lee, Brandon; Lee, Ryan; Levine, Kevin; Li, Yedda; Liu, Bo; Liu, Lucy; Liu, Max; Lousararian, Adam; Ma, Jimmy; Mallya, Allyson; Manchee, Charlie; Marcus, Joseph; McDaniel, Stephen; Miller, Michelle L; Molleston, Jerome M; Diez, Cristina Montero; Ng, Patrick; Ngai, Natalie; Nguyen, Hien; Nylander, Andrew; Pollack, Jason; Rastogi, Suchita; Reddy, Himabindu; Regenold, Nathaniel; Sarezky, Jon; Schultz, Michael; Shim, Jien; Skorupa, Tara; Smith, Kenneth; Spencer, Sarah J; Srikanth, Priya; Stancu, Gabriel; Stein, Andrew P; Strother, Marshall; Sudmeier, Lisa; Sun, Mengyang; Sundaram, Varun; Tazudeen, Noor; Tseng, Alan; Tzeng, Albert; Venkat, Rohit; Venkataram, Sandeep; Waldman, Leah; Wang, Tracy; Yang, Hao; Yu, Jack Y; Zheng, Yin; Preuss, Mary L; Garcia, Angelica; Juergens, Matt; Morris, Robert W; Nagengast, Alexis A; Azarewicz, Julie; Carr, Thomas J; Chichearo, Nicole; Colgan, Mike; Donegan, Megan; Gardner, Bob; Kolba, Nik; Krumm, Janice L; Lytle, Stacey; MacMillian, Laurell; Miller, Mary; Montgomery, Andrew; Moretti, Alysha; Offenbacker, Brittney; Polen, Mike; Toth, John; Woytanowski, John; Kadlec, Lisa; Crawford, Justin; Spratt, Mary L; Adams, Ashley L; Barnard, Brianna K; Cheramie, Martin N; Eime, Anne M; Golden, Kathryn L; Hawkins, Allyson P; Hill, Jessica E; Kampmeier, Jessica A; Kern, Cody D; Magnuson, Emily E; Miller, Ashley R; Morrow, Cody M; Peairs, Julia C; Pickett, Gentry L; Popelka, Sarah A; Scott, Alexis J; Teepe, Emily J; TerMeer, Katie A; Watchinski, Carmen A; Watson, Lucas A; Weber, Rachel E; Woodard, Kate A; Barnard, Daron C; Appiah, Isaac; Giddens, Michelle M; McNeil, Gerard P; Adebayo, Adeola; Bagaeva, Kate; Chinwong, Justina; Dol, Chrystel; George, Eunice; Haltaufderhyde, Kirk; Haye, Joanna; Kaur, Manpreet; Semon, Max; Serjanov, Dmitri; Toorie, Anika; Wilson, Christopher; Riddle, Nicole C; Buhler, Jeremy; Mardis, Elaine R; Elgin, Sarah C R

    2015-03-04

    The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25-50%) than euchromatic reference regions (3-11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11-27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4-3.6 vs. 8.4-8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu. Copyright © 2015 Leung et al.

  13. Combinatorial events of insertion sequences and ICE in Gram-negative bacteria.

    Science.gov (United States)

    Toleman, Mark A; Walsh, Timothy R

    2011-09-01

    The emergence of antibiotic and antimicrobial resistance in Gram-negative bacteria is incremental and linked to genetic elements that function in a so-called 'one-ended transposition' manner, including ISEcp1, ISCR elements and Tn3-like transposons. The power of these elements lies in their inability to consistently recognize one of their own terminal sequences, while recognizing more genetically distant surrogate sequences. This has the effect of mobilizing the DNA sequence found adjacent to their initial location. In general, resistance in Gram-negatives is closely linked to a few one-off events. These include the capture of the class 1 integron by a Tn5090-like transposon; the formation of the 3' conserved segment (3'-CS); and the fusion of the ISCR1 element to the 3'-CS. The structures formed by these rare events have been massively amplified and disseminated in Gram-negative bacteria, but hitherto, are rarely found in Gram-positives. Such events dominate current resistance gene acquisition and are instrumental in the construction of large resistance gene islands on chromosomes and plasmids. Similar combinatorial events appear to have occurred between conjugative plasmids and phages constructing hybrid elements called integrative and conjugative elements or conjugative transposons. These elements are beginning to be closely linked to some of the more powerful resistance mechanisms such as the extended spectrum β-lactamases, metallo- and AmpC type β-lactamases. Antibiotic resistance in Gram-negative bacteria is dominated by unusual combinatorial mistakes of Insertion sequences and gene fusions which have been selected and amplified by antibiotic pressure enabling the formation of extended resistance islands. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  14. Minos as a novel Tc1/mariner-type transposable element for functional genomic analysis in Aspergillus nidulans.

    Science.gov (United States)

    Evangelinos, Minoas; Anagnostopoulos, Gerasimos; Karvela-Kalogeraki, Iliana; Stathopoulou, Panagiota M; Scazzocchio, Claudio; Diallinas, George

    2015-08-01

    Transposons constitute powerful genetic tools for gene inactivation, exon or promoter trapping and genome analyses. The Minos element from Drosophila hydei, a Tc1/mariner-like transposon, has proved as a very efficient tool for heterologous transposition in several metazoa. In filamentous fungi, only a handful of fungal-specific transposable elements have been exploited as genetic tools, with the impala Tc1/mariner element from Fusarium oxysporum being the most successful. Here, we developed a two-component transposition system to manipulate Minos transposition in Aspergillus nidulans (AnMinos). Our system allows direct selection of transposition events based on re-activation of niaD, a gene necessary for growth on nitrate as a nitrogen source. On average, among 10(8) conidiospores, we obtain up to ∼0.8×10(2) transposition events leading to the expected revertant phenotype (niaD(+)), while ∼16% of excision events lead to AnMinos loss. Characterized excision footprints consisted of the four terminal bases of the transposon flanked by the TA target duplication and led to no major DNA rearrangements. AnMinos transposition depends on the presence of its homologous transposase. Its frequency was not significantly affected by temperature, UV irradiation or the transcription status of the original integration locus (niaD). Importantly, transposition is dependent on nkuA, encoding an enzyme essential for non-homologous end joining of DNA in double-strand break repair. AnMinos proved to be an efficient tool for functional analysis as it seems to transpose in different genomic loci positions in all chromosomes, including a high proportion of integration events within or close to genes. We have used Minos to obtain morphological and toxic analogue resistant mutants. Interestingly, among morphological mutants some seem to be due to Minos-elicited over-expression of specific genes, rather than gene inactivation. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Comparative analysis of the genomes of two field isolates of the rice blast fungus Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Minfeng Xue

    Full Text Available Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases of rice worldwide. The fungal pathogen is notorious for its ability to overcome host resistance. To better understand its genetic variation in nature, we sequenced the genomes of two field isolates, Y34 and P131. In comparison with the previously sequenced laboratory strain 70-15, both field isolates had a similar genome size but slightly more genes. Sequences from the field isolates were used to improve genome assembly and gene prediction of 70-15. Although the overall genome structure is similar, a number of gene families that are likely involved in plant-fungal interactions are expanded in the field isolates. Genome-wide analysis on asynonymous to synonymous nucleotide substitution rates revealed that many infection-related genes underwent diversifying selection. The field isolates also have hundreds of isolate-specific genes and a number of isolate-specific gene duplication events. Functional characterization of randomly selected isolate-specific genes revealed that they play diverse roles, some of which affect virulence. Furthermore, each genome contains thousands of loci of transposon-like elements, but less than 30% of them are conserved among different isolates, suggesting active transposition events in M. oryzae. A total of approximately 200 genes were disrupted in these three strains by transposable elements. Interestingly, transposon-like elements tend to be associated with isolate-specific or duplicated sequences. Overall, our results indicate that gain or loss of unique genes, DNA duplication, gene family expansion, and frequent translocation of transposon-like elements are important factors in genome variation of the rice blast fungus.

  16. LTR retrotransposons in fungi.

    Directory of Open Access Journals (Sweden)

    Anna Muszewska

    Full Text Available Transposable elements with long terminal direct repeats (LTR TEs are one of the best studied groups of mobile elements. They are ubiquitous elements present in almost all eukaryotic genomes. Their number and state of conservation can be a highlight of genome dynamics. We searched all published fungal genomes for LTR-containing retrotransposons, including both complete, functional elements and remnant copies. We identified a total of over 66,000 elements, all of which belong to the Ty1/Copia or Ty3/Gypsy superfamilies. Most of the detected Gypsy elements represent Chromoviridae, i.e. they carry a chromodomain in the pol ORF. We analyzed our data from a genome-ecology perspective, looking at the abundance of various types of LTR TEs in individual genomes and at the highest-copy element from each genome. The TE content is very variable among the analyzed genomes. Some genomes are very scarce in LTR TEs (8000 elements. The data shows that transposon expansions in fungi usually involve an increase both in the copy number of individual elements and in the number of element types. The majority of the highest-copy TEs from all genomes are Ty3/Gypsy transposons. Phylogenetic analysis of these elements suggests that TE expansions have appeared independently of each other, in distant genomes and at different taxonomical levels. We also analyzed the evolutionary relationships between protein domains encoded by the transposon pol ORF and we found that the protease is the fastest evolving domain whereas reverse transcriptase and RNase H evolve much slower and in correlation with each other.

  17. Domestication of transposable elements into MicroRNA genes in plants.

    Directory of Open Access Journals (Sweden)

    Yang Li

    Full Text Available Transposable elements (TE usually take up a substantial portion of eukaryotic genome. Activities of TEs can cause genome instability or gene mutations that are harmful or even disastrous to the host. TEs also contribute to gene and genome evolution at many aspects. Part of miRNA genes in mammals have been found to derive from transposons while convincing evidences are absent for plants. We found that a considerable number of previously annotated plant miRNAs are identical or homologous to transposons (TE-MIR, which include a small number of bona fide miRNA genes that conform to generally accepted plant miRNA annotation rules, and hairpin derived siRNAs likely to be pre-evolved miRNAs. Analysis of these TE-MIRs indicate that transitions from the medium to high copy TEs into miRNA genes may undergo steps such as inverted repeat formation, sequence speciation and adaptation to miRNA biogenesis. We also identified initial target genes of the TE-MIRs, which contain homologous sequences in their CDS as consequence of cognate TE insertions. About one-third of the initial target mRNAs are supported by publicly available degradome sequencing data for TE-MIR sRNA induced cleavages. Targets of the TE-MIRs are biased to non-TE related genes indicating their penchant to acquire cellular functions during evolution. Interestingly, most of these TE insertions span boundaries between coding and non-coding sequences indicating their incorporation into CDS through alteration of splicing or translation start or stop signals. Taken together, our findings suggest that TEs in gene rich regions can form foldbacks in non-coding part of transcripts that may eventually evolve into miRNA genes or be integrated into protein coding sequences to form potential targets in a "temperate" manner. Thus, transposons may supply as resources for the evolution of miRNA-target interactions in plants.

  18. Complete sequence of the IncA/C1 plasmid pCf587 carrying blaPER-2 from Citrobacter freundii.

    Science.gov (United States)

    Ruggiero, Melina; Girlich, Delphine; Dabos, Laura; Power, Pablo; Naas, Thierry; Gutkind, Gabriel

    2018-02-20

    The bla PER-2 harboring plasmid pCf587 (191,541 bp) belongs to lineage IncA/C 1 and is closely related to pRA1. It contains a large resistance island including the bla PER-2 gene between two copies of IS Kox2 -like elements, the toxin-antitoxin module pemK-pemI, several other resistance genes inserted within a Tn 2 transposon, a Tn 21 -like structure, and a class 1 integron. pCf587 belongs into ST 13, a new pMLST. Copyright © 2018 American Society for Microbiology.

  19. Comparison of 26 sphingomonad genomes reveals diverse environmental adaptations and biodegradative capabilities

    DEFF Research Database (Denmark)

    Aylward, Frank O.; McDonald, Bradon R.; Adams, Sandra M.

    2013-01-01

    to the genus Sphingobium. Our pan-genomic analysis of sphingomonads reveals numerous species-specific open reading frames (ORFs) but few signatures of genus-specific cores. The organization and coding potential of the sphingomonad genomes appear to be highly variable, and plasmid-mediated gene transfer...... and chromosome-plasmid recombination, together with prophage- and transposon-mediated rearrangements, appear to play prominent roles in the genome evolution of this group. We find that many of the sphingomonad genomes encode numerous oxygenases and glycoside hydrolases, which are likely responsible...... a basis for understanding the ecological strategies employed by sphingomonads and their role in environmental nutrient cycling....

  20. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

    DEFF Research Database (Denmark)

    Uren, Anthony G; Mikkers, Harald; Kool, Jaap

    2009-01-01

    sites has been a major limitation to performing screens on this scale. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA......Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion...

  1. SiRNAs in vivo imaging: methodology of fluorine-18 radiolabelling and application for the optimization of the siRNAs biodistribution and pharmaceutical properties

    International Nuclear Information System (INIS)

    Viel, Th.

    2008-01-01

    As RNA interference is a natural process which enables eukaryote cells to regulate the gene expressions, to control transposons, and to struggle against some viruses, two imagery techniques have been used in this research, i.e. optical imagery and Positron Emission Tomography (PET) imagery, to study the various modifications of the small interferential RNAs (siRNA). Different chemically modified siRNAs have been prepared and their in vitro activity, their in vivo metabolism (by HPLC analysis), their bio-distribution and their pharmacokinetic properties (by PET imagery) after marking them with fluorine-18. Their in vivo activity has been assessed by optical imagery

  2. Efficient Genome Editing in Induced Pluripotent Stem Cells with Engineered Nucleases In Vitro.

    Science.gov (United States)

    Termglinchan, Vittavat; Seeger, Timon; Chen, Caressa; Wu, Joseph C; Karakikes, Ioannis

    2017-01-01

    Precision genome engineering is rapidly advancing the application of the induced pluripotent stem cells (iPSCs) technology for in vitro disease modeling of cardiovascular diseases. Targeted genome editing using engineered nucleases is a powerful tool that allows for reverse genetics, genome engineering, and targeted transgene integration experiments to be performed in a precise and predictable manner. However, nuclease-mediated homologous recombination is an inefficient process. Herein, we describe the development of an optimized method combining site-specific nucleases and the piggyBac transposon system for "seamless" genome editing in pluripotent stem cells with high efficiency and fidelity in vitro.

  3. CpG + CpNpG Analysis of Protein-Coding Sequences from Tomato

    DEFF Research Database (Denmark)

    Hobolth, Asger; Nielsen, Rasmus; Wang, Ying

    2006-01-01

    We develop codon-based models for simultaneously inferring the mutational effects of CpG and CpNpG methylation in coding regions. In a data set of 369 tomato genes, we show that there is very little effect of CpNpG methylation but a strong effect of CpG methylation affecting almost all genes. We...... further show that the CpNpG and CpG effects are largely uncorrelated. Our results suggest different roles of CpG and CpNpG methylation, with CpNpG methylation possibly playing a specialized role in defense against transposons and RNA viruses....

  4. Genetic-Metabolic Coupling for Targeted Metabolic Engineering

    DEFF Research Database (Denmark)

    Cardinale, Stefano; Tueros Farfan, Felipe Gonzalo; Sommer, Morten Otto Alexander

    2017-01-01

    pertur-bations, thus limiting their relevance to specific conditions. Here, we address this issue by coupling cell fitness to the production of thiamine diphosphate in Escherichia coli using a synthetic RNA biosensor. We use this strategy to interrogate a library of transposon mutants and elucidate...... the native gene network influencing both cell fitness and thiamine production. Ultimately, we identify effectors of the OxyR-Fur stress response that limit thiamine biosynthesis via alternative regulation of iron storage and Fe-S cluster inclusion in enzymes. This study presents a new approach...

  5. Nitrogen regulates chitinase gene expression in a marine bacterium

    DEFF Research Database (Denmark)

    Delpin, Marina; Goodman, A.E.

    2009-01-01

    Ammonium concentration and nitrogen source regulate promoter activity and use for the transcription of chiA, the major chitinase gene of Pseudoalteromonas sp. S91 and S91CX, an S91 transposon lacZ fusion mutant. The activity of chiA was quantified by beta-galactosidase assay of S91CX cultures con...... GlcNAc, transcription initiated from two putative sigma(54)-dependent promoters and (3) glt, transcription initiated from all three putative promoters. The ISME Journal (2009) 3, 1064-1069; doi:10.1038/ismej.2009.49; published online 14 May 2009...

  6. Arabidopsis VARIEGATED 3 encodes a chloroplast-targeted, zinc-finger protein required for chloroplast and palisade cell development

    DEFF Research Database (Denmark)

    Næsted, Henrik; Holm, Agnethe; Jenkins, Tom

    2004-01-01

    The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3...... that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development....

  7. Arabidopsis VARIEGATED 3 encodes a chloroplasttargeted, zinc-finger protein required for chloroplast and palisade cell development

    DEFF Research Database (Denmark)

    Næsted, Henrik; Holm, A.; Jenkins, T.

    2004-01-01

    The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3...... that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development....

  8. Single-copy insertion of transgenes in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Frøkjaer-Jensen, Christian; Davis, M Wayne; Hopkins, Christopher E

    2008-01-01

    developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can...... be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines....

  9. Assaying gene function by growth competition experiment.

    Science.gov (United States)

    Merritt, Joshua; Edwards, Jeremy S

    2004-07-01

    High-throughput screening and analysis is one of the emerging paradigms in biotechnology. In particular, high-throughput methods are essential in the field of functional genomics because of the vast amount of data generated in recent and ongoing genome sequencing efforts. In this report we discuss integrated functional analysis methodologies which incorporate both a growth competition component and a highly parallel assay used to quantify results of the growth competition. Several applications of the two most widely used technologies in the field, i.e., transposon mutagenesis and deletion strain library growth competition, and individual applications of several developing or less widely reported technologies are presented.

  10. Identification of Mycobacterial Genes Involved in Antibiotic Sensitivity: Implications for the Treatment of Tuberculosis with β-Lactam-Containing Regimens

    Science.gov (United States)

    Viswanathan, Gopinath; Yadav, Sangya

    2017-01-01

    ABSTRACT In a Mycobacterium smegmatis mutant library screen, transposon mutants with insertions in fhaA, dprE2, rpsT, and parA displayed hypersusceptibility to antibiotics, including the β-lactams meropenem, ampicillin, amoxicillin, and cefotaxime. Sub-MIC levels of octoclothepin, a psychotic drug inhibiting ParA, phenocopied the parA insertion and enhanced the bactericidal activity of meropenem against Mycobacterium tuberculosis in combination with clavulanate. Our study identifies novel factors associated with antibiotic resistance, with implications in repurposing β-lactams for tuberculosis treatment. PMID:28438925

  11. Isolation and characterization of Salmonella typhimurium glyoxylate shunt mutants.

    OpenAIRE

    Wilson, R B; Maloy, S R

    1987-01-01

    Growth of Salmonella typhimurium on acetate as a sole carbon source requires expression of the glyoxylate shunt; however, the genes for the glyoxylate shunt enzymes have not been previously identified in S. typhimurium. In this study, we isolated transposon insertions in the genes for the two unique enzymes of this pathway, aceA (isocitrate lyase) and aceB (malate synthase). The aceA and aceB genes were located at 89.5 min on the S. typhimurium genetic map. Genetic linkage to nearby loci indi...

  12. Insertional inactivation of a chromosomal locus that modulates expression of potential virulence determinants in Staphylococcus aureus.

    OpenAIRE

    Cheung, A L; Wolz, C; Yeaman, M R; Bayer, A S

    1995-01-01

    A single insertion of transposon Tn551 into a unique chromosomal locus of Staphylococcus aureus ISP479C has resulted in a pleiotropic effect on the expression of both extracellular and cell wall proteins. In particular, the expression of cell wall protein A and clumping activity with fibrinogen were rendered undetectable in the mutant 1E3 compared with the parent. The secretion of alpha-hemolysin in mutant 1E3 was modestly increased. Southern blot and phenotypic analyses indicated that this l...

  13. Secretome discovery reveals lignocellulose degradation capacity of the ectomycorrhizal fungus Paxillus involutus

    DEFF Research Database (Denmark)

    Roth, Doris; Rineau, Francois; Olsen, Peter B.

    2011-01-01

    To improve our understanding of the role ectomycorrhizal fungi play in biomass conversion, we studied the transcriptome of P. involutus grown on glass beads in extract of soil organic matter. The mycelium was used for a cDNA library screened by Transposon-Assisted Signal Trapping (TAST*) for gene...... the brown rot fungi systems. In addition, GH61 apparently acts as accessory protein both in enzymatic and in radical-based cellulolysis. * Becker et al., J. Microbial Methods, 2004, 57(1), 123-33....

  14. An additional regulator, TsaQ, is involved with TsaR in regulation of transport during the degradation of p-toluenesulfonate in Comamonas testosteroni T-2

    OpenAIRE

    Tralau, Tewes; Cook, Alasdair M.; Ruff, Jürgen

    2003-01-01

    The degradation of p-toluenesulfonate (TSA) by Comamonas testosteroni T-2 is initiated by a transport system (TsaST) and enzymes (TsaMBCD) encoded on the tsa transposon, Tn tsa, on the TSA plasmid (pTSA). Tn tsa comprises an insert of 15 kb between two IS 1071 elements. The left-hand 6 kb and the right-hand 6 kb are nearly mirror images. The regulator of the tsaMBCD1 genes (right-hand side) is the centrally located LysR-type TsaR, which is encoded upstream of tsaMBCD1 on the reverse strand. T...

  15. Characterization of TsaR, an Oxygen-Sensitive LysR-Type Regulator for the Degradation of p-Toluenesulfonate in Comamonas testosteroni T-2

    OpenAIRE

    Tralau, Tewes; Mampel, Jörg; Cook, Alasdair M.; Ruff, Jürgen

    2003-01-01

    TsaR is the putative LysR-type regulator of the tsa operon (tsaMBCD) which encodes the first steps in the degradation of p-toluenesulfonate (TSA) in Comamonas testosteroni T-2. Transposon mutagenesis was used to knock out tsaR. The resulting mutant lacked the ability to grow with TSA and p-toluenecarboxylate (TCA). Reintroduction of tsaR in trans on an expression vector reconstituted growth with TSA and TCA. The tsaR gene was cloned into Escherichia coli with a C-terminal His tag and overexpr...

  16. Endogenous reverse transcriptase (RT) activity and Chromatin remodeling in normal and transformed cells and early embryos

    International Nuclear Information System (INIS)

    Spadafora, C.; Sciamanna, I.; Misteli, T.

    2009-01-01

    Endogenous Reverse Transcriptase (RT) is an enzyme encoded by two classes of genomic retro-elements: retro-transposons and endogenous retroviruses. Basal levels of RT are expressed in all non pathological, differentiated tissues while high RT expression levels characterize tumorigenic cells, germ cells and embryonic tissues. Preliminary studies carried out in our laboratory have shown that RT inhibition using pharmacological inhibitors (nevirapine and efavirenz, two drugs currently used in AIDS therapy) drastically reduces cell proliferation, promotes differentiation of tumorigenic cells in vitro, induces a reprogrammed gene expression and antagonizes tumor progression in nude mice inoculated with tumorigenic human cell lines, including melanoma, prostate and colon carcinoma and microcitoma

  17. SiRNAs in vivo imaging: methodology of fluorine-18 radiolabelling and application for the optimization of the siRNAs biodistribution and pharmaceutical properties; Imagerie in vivo des ARN interferentiels: methodologie de marquage au fluor-18 et application pour l'optimisation par imagerie de leur biodistribution et de leurs proprietes pharmacologiques

    Energy Technology Data Exchange (ETDEWEB)

    Viel, Th

    2008-01-15

    As RNA interference is a natural process which enables eukaryote cells to regulate the gene expressions, to control transposons, and to struggle against some viruses, two imagery techniques have been used in this research, i.e. optical imagery and Positron Emission Tomography (PET) imagery, to study the various modifications of the small interferential RNAs (siRNA). Different chemically modified siRNAs have been prepared and their in vitro activity, their in vivo metabolism (by HPLC analysis), their bio-distribution and their pharmacokinetic properties (by PET imagery) after marking them with fluorine-18. Their in vivo activity has been assessed by optical imagery.

  18. The secondary resistome of multidrug-resistant Klebsiella pneumoniae

    DEFF Research Database (Denmark)

    Jana, Bimal; Cain, Amy K.; Doerrler, William T.

    2017-01-01

    insertions and measured mutant depletion upon exposure to three clinically relevant antimicrobials (colistin, imipenem or ciprofloxacin) by Transposon Directed Insertion-site Sequencing (TraDIS). Using this high-throughput approach, we defined three sets of chromosomal non-essential genes essential...... for growth during exposure to colistin (n = 35), imipenem (n = 1) or ciprofloxacin (n = 1) in addition to known resistance determinants, collectively termed the “secondary resistome”. As proof of principle, we demonstrated that inactivation of a non-essential gene not previously found linked to colistin...

  19. Long interspersed element-1 (LINE-1): passenger or driver in human neoplasms?

    Science.gov (United States)

    Rodić, Nemanja; Burns, Kathleen H

    2013-03-01

    LINE-1 (L1) retrotransposons make up a significant portion of human genomes, with an estimated 500,000 copies per genome. Like other retrotransposons, L1 retrotransposons propagate through RNA sequences that are reverse transcribed into DNA sequences, which are integrated into new genomic loci. L1 somatic insertions have the potential to disrupt the transcriptome by inserting into or nearby genes. By mutating genes and playing a role in epigenetic dysregulation, L1 transposons may contribute to tumorigenesis. Studies of the "mobilome" have lagged behind other tumor characterizations at the sequence, transcript, and epigenetic levels. Here, we consider evidence that L1 retrotransposons may sometimes drive human tumorigenesis.

  20. An abyssal mobilome: viruses, plasmids and vesicles from deep-sea hydrothermal vents.

    Science.gov (United States)

    Lossouarn, Julien; Dupont, Samuel; Gorlas, Aurore; Mercier, Coraline; Bienvenu, Nadege; Marguet, Evelyne; Forterre, Patrick; Geslin, Claire

    2015-12-01

    Mobile genetic elements (MGEs) such as viruses, plasmids, vesicles, gene transfer agents (GTAs), transposons and transpovirions, which collectively represent the mobilome, interact with cellular organisms from all three domains of life, including those thriving in the most extreme environments. While efforts have been made to better understand deep-sea vent microbial ecology, our knowledge of the mobilome associated with prokaryotes inhabiting deep-sea hydrothermal vents remains limited. Here we focus on the abyssal mobilome by reviewing accumulating data on viruses, plasmids and vesicles associated with thermophilic and hyperthermophilic Bacteria and Archaea present in deep-sea hydrothermal vents. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  1. Long interspersed element-1 (LINE-1: passenger or driver in human neoplasms?

    Directory of Open Access Journals (Sweden)

    Nemanja Rodić

    2013-03-01

    Full Text Available LINE-1 (L1 retrotransposons make up a significant portion of human genomes, with an estimated 500,000 copies per genome. Like other retrotransposons, L1 retrotransposons propagate through RNA sequences that are reverse transcribed into DNA sequences, which are integrated into new genomic loci. L1 somatic insertions have the potential to disrupt the transcriptome by inserting into or nearby genes. By mutating genes and playing a role in epigenetic dysregulation, L1 transposons may contribute to tumorigenesis. Studies of the "mobilome" have lagged behind other tumor characterizations at the sequence, transcript, and epigenetic levels. Here, we consider evidence that L1 retrotransposons may sometimes drive human tumorigenesis.

  2. Isolation and sequence analysis of the wheat B genome subtelomeric DNA

    Directory of Open Access Journals (Sweden)

    Huneau Cecile

    2009-09-01

    Full Text Available Abstract Background Telomeric and subtelomeric regions are essential for genome stability and regular chromosome replication. In this work, we have characterized the wheat BAC (bacterial artificial chromosome clones containing Spelt1 and Spelt52 sequences, which belong to the subtelomeric repeats of the B/G genomes of wheats and Aegilops species from the section Sitopsis. Results The BAC library from Triticum aestivum cv. Renan was screened using Spelt1 and Spelt52 as probes. Nine positive clones were isolated; of them, clone 2050O8 was localized mainly to the distal parts of wheat chromosomes by in situ hybridization. The distribution of the other clones indicated the presence of different types of repetitive sequences in BACs. Use of different approaches allowed us to prove that seven of the nine isolated clones belonged to the subtelomeric chromosomal regions. Clone 2050O8 was sequenced and its sequence of 119 737 bp was annotated. It is composed of 33% transposable elements (TEs, 8.2% Spelt52 (namely, the subfamily Spelt52.2 and five non-TE-related genes. DNA transposons are predominant, making up 24.6% of the entire BAC clone, whereas retroelements account for 8.4% of the clone length. The full-length CACTA transposon Caspar covers 11 666 bp, encoding a transposase and CTG-2 proteins, and this transposon accounts for 40% of the DNA transposons. The in situ hybridization data for 2050O8 derived subclones in combination with the BLAST search against wheat mapped ESTs (expressed sequence tags suggest that clone 2050O8 is located in the terminal bin 4BL-10 (0.95-1.0. Additionally, four of the predicted 2050O8 genes showed significant homology to four putative orthologous rice genes in the distal part of rice chromosome 3S and confirm the synteny to wheat 4BL. Conclusion Satellite DNA sequences from the subtelomeric regions of diploid wheat progenitor can be used for selecting the BAC clones from the corresponding regions of hexaploid wheat

  3. Isolation and sequence analysis of the wheat B genome subtelomeric DNA.

    Science.gov (United States)

    Salina, Elena A; Sergeeva, Ekaterina M; Adonina, Irina G; Shcherban, Andrey B; Afonnikov, Dmitry A; Belcram, Harry; Huneau, Cecile; Chalhoub, Boulos

    2009-09-05

    Telomeric and subtelomeric regions are essential for genome stability and regular chromosome replication. In this work, we have characterized the wheat BAC (bacterial artificial chromosome) clones containing Spelt1 and Spelt52 sequences, which belong to the subtelomeric repeats of the B/G genomes of wheats and Aegilops species from the section Sitopsis. The BAC library from Triticum aestivum cv. Renan was screened using Spelt1 and Spelt52 as probes. Nine positive clones were isolated; of them, clone 2050O8 was localized mainly to the distal parts of wheat chromosomes by in situ hybridization. The distribution of the other clones indicated the presence of different types of repetitive sequences in BACs. Use of different approaches allowed us to prove that seven of the nine isolated clones belonged to the subtelomeric chromosomal regions. Clone 2050O8 was sequenced and its sequence of 119,737 bp was annotated. It is composed of 33% transposable elements (TEs), 8.2% Spelt52 (namely, the subfamily Spelt52.2) and five non-TE-related genes. DNA transposons are predominant, making up 24.6% of the entire BAC clone, whereas retroelements account for 8.4% of the clone length. The full-length CACTA transposon Caspar covers 11,666 bp, encoding a transposase and CTG-2 proteins, and this transposon accounts for 40% of the DNA transposons. The in situ hybridization data for 2050O8 derived subclones in combination with the BLAST search against wheat mapped ESTs (expressed sequence tags) suggest that clone 2050O8 is located in the terminal bin 4BL-10 (0.95-1.0). Additionally, four of the predicted 2050O8 genes showed significant homology to four putative orthologous rice genes in the distal part of rice chromosome 3S and confirm the synteny to wheat 4BL. Satellite DNA sequences from the subtelomeric regions of diploid wheat progenitor can be used for selecting the BAC clones from the corresponding regions of hexaploid wheat chromosomes. It has been demonstrated for the first time

  4. A New Generalizable Test for Detection of Mutations Affecting Tn10 Transposition

    OpenAIRE

    Huisman, Olivier; Kleckner, Nancy

    1987-01-01

    We describe here a new rapid screen that allows easy detection of transposon or host mutations that affect Tn10 transposition in Escherichia coli. This test involves a new Tn10 derivative called the "mini-lacZ-kanR fusion hopper" or mini-Tn10-LK for short. This element does not direct expression of β-galactosidase when present at its original starting location on a suitably engineered plasmid or phage genome because it lacks appropriate transcription and translation start signals. However, t...

  5. Epigenetic control of mobile DNA as an interface between experience and genome change

    Directory of Open Access Journals (Sweden)

    James A. Shapiro

    2014-04-01

    Full Text Available Mobile DNA in the genome is subject to RNA-targeted epigenetic control. This control regulates the activity of transposons, retrotransposons and genomic proviruses. Many different life history experiences alter the activities of mobile DNA and the expression of genetic loci regulated by nearby insertions. The same experiences induce alterations in epigenetic formatting and lead to trans-generational modifications of genome expression and stability. These observations lead to the hypothesis that epigenetic formatting directed by non-coding RNA provides a molecular interface between life history events and genome alteration.

  6. Electrotransformation and clonal isolation of Rickettsia species

    Science.gov (United States)

    Riley, Sean P; Macaluso, Kevin R; Martinez, Juan J

    2015-01-01

    Genetic manipulation of obligate intracellular bacteria of the genus Rickettsia is currently undergoing a rapid period of change. The development of viable genetic tools, including replicative plasmids, transposons, homologous recombination, fluorescent protein-encoding genes, and antibiotic selectable markers has provided the impetus for future research development. This unit is designed to coalesce the basic methods pertaining to creation of genetically modified Rickettsia. The unit describes a series of methods, from inserting exogenous DNA into Rickettsia to the final isolation of genetically modified bacterial clones. Researchers working towards genetic manipulation of Rickettsia or similar obligate intracellular bacteria will find these protocols to be a valuable reference. PMID:26528784

  7. Formation of ethanol from lactose by Zymomonas mobilis

    Energy Technology Data Exchange (ETDEWEB)

    Goodman, A E; Strzelecki, A T; Rogers, P L

    1984-01-01

    The lactose transposon Tn951 on the IncP plasmid RP1, was introduced into a strain of Zymomonas mobilis. The lac Z gene was expressed and US -galactosidase was produced. The synthesis of US -galactosidase was induced by either isopropyl-US -D-thiogalactopyranoside or lactose, and was not sensitive to catabolite repression. Cells of Zymomonas mobilis containing Tn951 were unable to form colonies on lactose plates, but in liquid medium produced ethanol from lactose as the sole carbon source. The original strain of Zymomonas mobilis without Tn951 produced little or no US -galactosidase and was unable to produce ethanol from lactose in liquid medium.

  8. pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes

    Directory of Open Access Journals (Sweden)

    Arévalo-Rodríguez Miguel

    2011-02-01

    Full Text Available Abstract Background Since publication in 1977 of plasmid pBR322, many breakthroughs in Biology have depended on increasingly sophisticated vector platforms for analysis and engineering of given bacterial strains. Although restriction sites impose a certain format in the procedures for assembling cloned genes, every attempt thus far to standardize vector architecture and nomenclature has ended up in failure. While this state of affairs may still be tolerable for traditional one-at-a-time studies of single genes, the onset of systems and synthetic biology calls for a simplification -along with an optimization- of the currently unwieldy pool of genetic tools. Results The functional DNA sequences present in the natural bacterial transposon Tn5 have been methodically edited and refactored for the production of a multi-purpose genetic tool named pBAM1, which allows a range of manipulations in the genome of Gram-negative bacteria. This all-synthetic construct enhances the power of mini-transposon vectors for either de-construction or re-construction of phenotypes á la carte by incorporating features inspired in systems engineering: modularity, re-usability, minimization, and compatibility with other genetic tools. pBAM1 bears an streamlined, restriction site-freed and narrow-host range replication frame bearing the sequences of R6K oriV, oriT and an ampicillin resistance marker. These go along with a business module that contains a host-independent and hyperactive transposition platform for in vivo or in vitro insertion of desired DNA into the genome of the target bacterium. All functional sequences were standardized for a straightforward replacement by equivalent counterparts, if required. pBAM1 can be delivered into recipient cells by either mating or electroporation, producing transposon insertion frequencies of 1.8 × 10-3 and 1.02 × 10-7, respectively in the soil bacterium Pseudomonas putida. Analyses of the resulting clones revealed a 100% of

  9. A Pseudomonas putida strain genetically engineered for 1,2,3-trichloropropane bioremediation.

    Science.gov (United States)

    Samin, Ghufrana; Pavlova, Martina; Arif, M Irfan; Postema, Christiaan P; Damborsky, Jiri; Janssen, Dick B

    2014-09-01

    1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3-dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCP-degrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP-degrading bacterium Pseudomonas putida MC4. For this purpose, the dehalogenase gene (dhaA31) was cloned behind the constitutive dhlA promoter and was introduced into the genome of strain MC4 using a transposon delivery system. The transposon-located antibiotic resistance marker was subsequently removed using a resolvase step. Growth of the resulting engineered bacterium, P. putida MC4-5222, on TCP was indeed observed, and all organic chlorine was released as chloride. A packed-bed reactor with immobilized cells of strain MC4-5222 degraded >95% of influent TCP (0.33 mM) under continuous-flow conditions, with stoichiometric release of inorganic chloride. The results demonstrate the successful use of a laboratory-evolved dehalogenase and genetic engineering to produce an effective, plasmid-free, and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated hydrocarbon. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  10. Autolysis of methicillin-resistant and -susceptible Staphylococcus aureus.

    Science.gov (United States)

    Gustafson, J E; Berger-Bächi, B; Strässle, A; Wilkinson, B J

    1992-01-01

    The autolytic activities, including unstimulated, Triton X-100-stimulated, and daptomycin-induced, of various sets of methicillin-resistant and related methicillin-susceptible strains were compared. Faster rates of autolysis were noted in two heterogeneous methicillin-resistant transductants than in their methicillin-susceptible parental recipients, in a heterogeneous resistant strain than in a susceptible derivative created by chemical mutagenesis, and in a homogeneous resistant strain than in a derivative that had decreased methicillin resistance and was created by transposon Tn551 mutagenesis. These results suggest that the presence of the methicillin resistance region, mec, either directly or indirectly through an interaction with other host genes, confers a faster rate of autolysis on strains. Various auxilliary genes are known to affect methicillin resistance expression, and one of these genes, femA, was necessary for the expression of this faster rate of autolysis. These differences in autolytic activities were not observed in isolated crude cell walls retaining autolytic activities, suggesting different modes of regulation of autolysins in intact cells and isolated walls. In contrast, one homogeneous, highly resistant strain, DU4916, had a lower autolytic activity than did derived heterogeneous resistant and susceptible strains created by chemical mutagenesis and a strain that had decreased resistance and was created by transposon mutagenesis. Our observations suggest that methicillin resistance expression is associated with an enhanced rate of autolysis, in heterogeneous resistant strains at least. Images PMID:1320363

  11. Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments.

    Science.gov (United States)

    Miller, Daniel P; Hutcherson, Justin A; Wang, Yan; Nowakowska, Zuzanna M; Potempa, Jan; Yoder-Himes, Deborah R; Scott, David A; Whiteley, Marvin; Lamont, Richard J

    2017-01-01

    Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis , and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism.

  12. Whole genome resequencing reveals natural target site preferences of transposable elements in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Raquel S Linheiro

    Full Text Available Transposable elements are mobile DNA sequences that integrate into host genomes using diverse mechanisms with varying degrees of target site specificity. While the target site preferences of some engineered transposable elements are well studied, the natural target preferences of most transposable elements are poorly characterized. Using population genomic resequencing data from 166 strains of Drosophila melanogaster, we identified over 8,000 new insertion sites not present in the reference genome sequence that we used to decode the natural target preferences of 22 families of transposable element in this species. We found that terminal inverted repeat transposon and long terminal repeat retrotransposon families present clade-specific target site duplications and target site sequence motifs. Additionally, we found that the sequence motifs at transposable element target sites are always palindromes that extend beyond the target site duplication. Our results demonstrate the utility of population genomics data for high-throughput inference of transposable element targeting preferences in the wild and establish general rules for terminal inverted repeat transposon and long terminal repeat retrotransposon target site selection in eukaryotic genomes.

  13. Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits.

    Directory of Open Access Journals (Sweden)

    Andrea Kerekes

    Full Text Available Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals.

  14. A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition in rice

    KAUST Repository

    La, Honggui; Ding, Bo; Mishra, Gyan Prakash; Zhou, Bo; Yang, Hongmei; Bellizzi, Maria Del Rosario; Chen, Songbiao; Meyers, Blake C.; Peng, Zhaohua; Zhu, Jian-Kang; Wang, Guoliang

    2011-01-01

    DNA 5-methylcytosine (5-meC) is an important epigenetic mark for transcriptional gene silencing in many eukaryotes. In Arabidopsis, 5-meC DNA glycosylase/lyases actively remove 5-meC to counter-act transcriptional gene silencing in a locus-specific manner, and have been suggested to maintain the expression of transposons. However, it is unclear whether plant DNA demethylases can promote the transposition of transposons. Here we report the functional characterization of the DNA glycosylase/lyase DNG701 in rice. DNG701 encodes a large (1,812 amino acid residues) DNA glycosylase domain protein. Recombinant DNG701 protein showed 5-meC DNA glycosylase and lyase activities in vitro. Knockout or knockdown of DNG701 in rice plants led to DNA hypermethylation and reduced expression of the retrotransposon Tos17. Tos17 showed less transposition in calli derived from dng701 knockout mutant seeds compared with that in wild-type calli. Overexpression of DNG701 in both rice calli and transgenic plants substantially reduced DNA methylation levels of Tos17 and enhanced its expression. The overexpression also led to more frequent transposition of Tos17 in calli. Our results demonstrate that rice DNG701 is a 5-meC DNA glycosylase/lyase responsible for the demethylation of Tos17 and this DNA demethylase plays a critical role in promoting Tos17 transposition in rice calli.

  15. Regulation of hemolysin expression and virulence of Staphylococcus aureus by a serine/threonine kinase and phosphatase.

    Directory of Open Access Journals (Sweden)

    Kellie Burnside

    2010-06-01

    Full Text Available Exotoxins, including the hemolysins known as the alpha (alpha and beta (beta toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1 were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1 increased expression. Transcription of the hla gene encoding alpha toxin was decreased in a Deltastp1 mutant strain and increased in a Deltastk1 strain. Microarray analysis of a Deltastk1 mutant revealed increased transcription of additional exotoxins. A Deltastp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Deltastk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU, serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE and a hypothetical protein (NWMN_1123 were present in the wild type and not in the Deltastk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.

  16. A PiggyBac mediated approach for lactoferricin gene transfer in bovine mammary epithelial stem cells for management of bovine mastitis.

    Science.gov (United States)

    Sharma, Neelesh; Huynh, Do Luong; Kim, Sung Woo; Ghosh, Mrinmoy; Sodhi, Simrinder Singh; Singh, Amit Kumar; Kim, Nam Eun; Lee, Sung Jin; Hussain, Kafil; Oh, Sung Jong; Jeong, Dong Kee

    2017-11-28

    The antibacterial and anti-inflammatory properties of lactoferricin have been ascribed to its ability to sequester essential iron. The objective of the study was to clone bovine lactoferricin ( LFcinB ) gene into PiggyBac Transposon vector, expression study in the bovine mammary epithelial stem cells (bMESCs) and also to determine the antimicrobial property of recombinant LFcinB against bovine mastitis-causing organisms. The PiggyBac-LFcinB was transfected into bMESCs by electroporation and a three fold of LFcinB secretion was observed in the transfected bMESCs medium by ELISA assay. Furthermore, the assessment of antimicrobial activity against mastitis causing pathogens Staphylococcus aureus and Escherichia coli demonstrated convincing evidence to prove strong antibacterial activity of LFcinB with 14.0±1.0 mm and 18.0±1.5 mm zone of inhibition against both organisms, respectively. The present study provides the convincing evidence to suggest the potential of PiggyBac transposon system to transfer antibacterial peptide into bMESCs or cow mammary gland and also pave the way to use bovine mammary gland as the bioreactors. Simultaneously, it also suggest toward commercial utilization of LFcinB bioreactor system in pharmaceutical industry.

  17. Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA.

    Science.gov (United States)

    Frothingham, R; Meeker-O'Connell, W A; Talbot, E A; George, J W; Kreuzer, K N

    1996-06-01

    Pyrazinamide (PZA) is one of the three most important drugs for treatment of Mycobacterium tuberculosis infections. The antibacterial activity of PZA requires a bacterial enzyme, pyrazinamidase (PZAase), which hydrolyzes PZA to form pyrazinoic acid and ammonia. Most PZA-resistant clinical M. tuberculosis isolates lack PZAase activity. With the goal of eventually identifying and characterizing the M.tuberculosis PZAase gene, we began with the more tractable organism, Escherichia coli, which also has PZAase activity. We screened a transposon-generated E. coli insertion mutant library, using a qualitative PZAase assay. Two PZAase-negative mutants out of 4,000 colonies screened were identified. In each mutant, the transposon interrupted the same 639-bp open reading frame (ORF), ORF1. The expression of ORF1 on a multicopy plasmid complemented a PZAase-negative mutant, leading to PZAase activity levels approximately 10-fold greater than those of the wild type. PZA has a structure similar to that of nicotinamide, a pyridine nucleotide cycle intermediate, so we tested our strains for nicotinamidase activity (EC 3.5.1.19) (genetic locus pncA). The construct with multiple plasmid copies of ORF1 had an approximately 10-fold increase in levels of nicotinamidase activity. This overexpressing strain could utilize nicotinamide as a sole nitrogen source, through wild-type E. coli cannot. We conclude that a single E. coli enzyme accounts for both PZAase and nicotinamidase activities and that ORF1 is the E.coli PZAase and nicotinamidase gene, pncA.

  18. Evolutionary origin of Rosaceae-specific active non-autonomous hAT elements and their contribution to gene regulation and genomic structural variation.

    Science.gov (United States)

    Wang, Lu; Peng, Qian; Zhao, Jianbo; Ren, Fei; Zhou, Hui; Wang, Wei; Liao, Liao; Owiti, Albert; Jiang, Quan; Han, Yuepeng

    2016-05-01

    Transposable elements account for approximately 30 % of the Prunus genome; however, their evolutionary origin and functionality remain largely unclear. In this study, we identified a hAT transposon family, termed Moshan, in Prunus. The Moshan elements consist of three types, aMoshan, tMoshan, and mMoshan. The aMoshan and tMoshan types contain intact or truncated transposase genes, respectively, while the mMoshan type is miniature inverted-repeat transposable element (MITE). The Moshan transposons are unique to Rosaceae, and the copy numbers of different Moshan types are significantly correlated. Sequence homology analysis reveals that the mMoshan MITEs are direct deletion derivatives of the tMoshan progenitors, and one kind of mMoshan containing a MuDR-derived fragment were amplified predominately in the peach genome. The mMoshan sequences contain cis-regulatory elements that can enhance gene expression up to 100-fold. The mMoshan MITEs can serve as potential sources of micro and long noncoding RNAs. Whole-genome re-sequencing analysis indicates that mMoshan elements are highly active, and an insertion into S-haplotype-specific F-box gene was reported to cause the breakdown of self-incompatibility in sour cherry. Taken together, all these results suggest that the mMoshan elements play important roles in regulating gene expression and driving genomic structural variation in Prunus.

  19. Unique attributes of cyanobacterial metabolism revealed by improved genome-scale metabolic modeling and essential gene analysis

    Science.gov (United States)

    Broddrick, Jared T.; Rubin, Benjamin E.; Welkie, David G.; Du, Niu; Mih, Nathan; Diamond, Spencer; Lee, Jenny J.; Golden, Susan S.; Palsson, Bernhard O.

    2016-01-01

    The model cyanobacterium, Synechococcus elongatus PCC 7942, is a genetically tractable obligate phototroph that is being developed for the bioproduction of high-value chemicals. Genome-scale models (GEMs) have been successfully used to assess and engineer cellular metabolism; however, GEMs of phototrophic metabolism have been limited by the lack of experimental datasets for model validation and the challenges of incorporating photon uptake. Here, we develop a GEM of metabolism in S. elongatus using random barcode transposon site sequencing (RB-TnSeq) essential gene and physiological data specific to photoautotrophic metabolism. The model explicitly describes photon absorption and accounts for shading, resulting in the characteristic linear growth curve of photoautotrophs. GEM predictions of gene essentiality were compared with data obtained from recent dense-transposon mutagenesis experiments. This dataset allowed major improvements to the accuracy of the model. Furthermore, discrepancies between GEM predictions and the in vivo dataset revealed biological characteristics, such as the importance of a truncated, linear TCA pathway, low flux toward amino acid synthesis from photorespiration, and knowledge gaps within nucleotide metabolism. Coupling of strong experimental support and photoautotrophic modeling methods thus resulted in a highly accurate model of S. elongatus metabolism that highlights previously unknown areas of S. elongatus biology. PMID:27911809

  20. Identification of a mutant locus that bypasses the BsgA protease requirement for social development in Myxococcus xanthus.

    Science.gov (United States)

    Cusick, John K; Hager, Elizabeth; Gill, Ronald E

    2015-01-01

    The BsgA protease is required for the earliest morphological changes observed in Myxococcus xanthus development. We hypothesize that the BsgA protease is required to cleave an inhibitor of the developmental program, and isolation of genetic bypass suppressors of a bsgA mutant was used to identify signaling components controlling development downstream of the BsgA protease. Strain M955 was created by transposon mutagenesis of a bsgA mutant followed by screening for strains that could develop despite the absence of the BsgA protease. Strain M955 was able to aggregate, form fruiting bodies, and partially restored the production of viable spores in comparison to the parental bsgA mutant. The bsgA Tn5Ω955 strain partially restored developmental expression to a subset of genes normally induced during development, and expressed one developmentally induced fusion at higher amounts during vegetative growth in comparison to wild-type cells. The transposon in strain M955 was localized to a Ribonuclease D homolog that appears to exist in an operon with a downstream aminopeptidase-encoding gene. The identification of a third distinct bypass suppressor of the BsgA protease suggests that the BsgA protease may regulate a potentially complex pathway during the initiation of the M. xanthus developmental program. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. An all-in-one, Tet-On 3G inducible PiggyBac system for human pluripotent stem cells and derivatives.

    Science.gov (United States)

    Randolph, Lauren N; Bao, Xiaoping; Zhou, Chikai; Lian, Xiaojun

    2017-05-08

    Human pluripotent stem cells (hPSCs) offer tremendous promise in tissue engineering and cell-based therapies due to their unique combination of two properties: pluripotency and unlimited proliferative capacity. However, directed differentiation of hPSCs to clinically relevant cell lineages is needed to achieve the goal of hPSC-based therapies. This requires a deep understanding of how cell signaling pathways converge on the nucleus to control differentiation and the ability to dissect gene function in a temporal manner. Here, we report the use of the PiggyBac transposon and a Tet-On 3G drug-inducible gene expression system to achieve versatile inducible gene expression in hPSC lines. Our new system, XLone, offers improvement over previous Tet-On systems with significantly reduced background expression and increased sensitivity to doxycycline. Transgene expression in hPSCs is tightly regulated in response to doxycycline treatment. In addition, the PiggyBac elements in our XLone construct provide a rapid and efficient strategy for generating stable transgenic hPSCs. Our inducible gene expression PiggyBac transposon system should facilitate the study of gene function and directed differentiation in human stem cells.

  2. Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats☆

    Science.gov (United States)

    Marzo, Mar; Liu, Danxu; Ruiz, Alfredo; Chalmers, Ronald

    2013-01-01

    Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity. PMID:23648487

  3. Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats.

    Science.gov (United States)

    Marzo, Mar; Liu, Danxu; Ruiz, Alfredo; Chalmers, Ronald

    2013-08-01

    Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Genetic engineering including superseding microinjection: new ways to make GM pigs.

    Science.gov (United States)

    Galli, Cesare; Perota, Andrea; Brunetti, Dario; Lagutina, Irina; Lazzari, Giovanna; Lucchini, Franco

    2010-01-01

    Techniques for genetic engineering of swine are providing genetically modified animals of importance for the field of xenotransplantation, animal models for human diseases and for a variety of research applications. Many of these modifications have been directed toward avoiding naturally existing cellular and antibody responses to species-specific antigens. A number of techniques are today available to engineering the genome of mammals, these range from the well established less efficient method of DNA microinjection into the zygote, the use of viral vectors, to the more recent use of somatic cell nuclear transfer. The use of enzymatic engineering that are being developed now will refine the precision of the genetic modification combined with the use of new vectors like transposons. The use of somatic cell nuclear transfer is currently the most efficient way to generate genetically modified pigs. The development of enzymatic engineering with zinc-finger nucleases, recombinases and transposons will revolutionize the field. Nevertheless, genetic engineering in large domesticated animals will remain a challenging task. Recent improvements in several fields of cell and molecular biology offer new promises and opportunities toward an easier, cost-effective and efficient generation of transgenic pigs. © 2010 John Wiley & Sons A/S.

  5. Nucleotide sequence of pOLA52: a conjugative IncX1 plasmid from Escherichia coli which enables biofilm formation and multidrug efflux

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars H.; She, Qunxin

    2008-01-01

    . The plasmid was also classified as IncX1 with incompatibility testing. The conjugal transfer and plasmid maintenance regions of pOLA52 therefore seem to represent IncX1 orthologues of the well-characterized IncX2 plasmid R6K. Sequence homology searches in GenBank also suggested a considerably higher...... of type 3 fimbriae (mrkABCDF). The plasmid was found to be 51,602 bp long with 68 putative genes. About half of the plasmid constituted a conserved IncX1-type backbone with predicted regions for conjugation, replication and partitioning, as well as a toxin/antitoxin (TA) plasmid addiction system...... prevalence of IncX1 group plasmids than IncX2. The 21 kb 'genetic load' region of pOLA52 was shown to consist of a mosaic, among other things a fragmented Tn3 transposon encoding ampicillin resistance. Most notably the oqxAB and mrkABCDF cassettes were contained within two composite transposons (Tn6010...

  6. Comparative analysis of pepper and tomato reveals euchromatin expansion of pepper genome caused by differential accumulation of Ty3/Gypsy-like elements

    Directory of Open Access Journals (Sweden)

    Ahn Jong Hwa

    2011-01-01

    Full Text Available Abstract Background Among the Solanaceae plants, the pepper genome is three times larger than that of tomato. Although the gene repertoire and gene order of both species are well conserved, the cause of the genome-size difference is not known. To determine the causes for the expansion of pepper euchromatic regions, we compared the pepper genome to that of tomato. Results For sequence-level analysis, we generated 35.6 Mb of pepper genomic sequences from euchromatin enriched 1,245 pepper BAC clones. The comparative analysis of orthologous gene-rich regions between both species revealed insertion of transposons exclusively in the pepper sequences, maintaining the gene order and content. The most common type of the transposon found was the LTR retrotransposon. Phylogenetic comparison of the LTR retrotransposons revealed that two groups of Ty3/Gypsy-like elements (Tat and Athila were overly accumulated in the pepper genome. The FISH analysis of the pepper Tat elements showed a random distribution in heterochromatic and euchromatic regions, whereas the tomato Tat elements showed heterochromatin-preferential accumulation. Conclusions Compared to tomato pepper euchromatin doubled its size by differential accumulation of a specific group of Ty3/Gypsy-like elements. Our results could provide an insight on the mechanism of genome evolution in the Solanaceae family.

  7. An Ancient Transcription Factor Initiates the Burst of piRNA Production During Early Meiosis in Mouse Testes

    Science.gov (United States)

    Li, Xin Zhiguo; Roy, Christian K.; Dong, Xianjun; Bolcun-Filas, Ewelina; Wang, Jie; Han, Bo W.; Xu, Jia; Moore, Melissa J.; Schimenti, John C.; Weng, Zhiping; Zamore, Phillip D.

    2013-01-01

    SUMMARY Animal germ cells produce PIWI-interacting RNAs (piRNAs), small silencing RNAs that suppress transposons and enable gamete maturation. Mammalian transposon-silencing piRNAs accumulate early in spermatogenesis, whereas pachytene piRNAs are produced later during post-natal spermatogenesis and account for >95% of all piRNAs in the adult mouse testis. Mutants defective for pachytene piRNA pathway proteins fail to produce mature sperm, but neither the piRNA precursor transcripts nor the trigger for pachytene piRNA production is known. Here, we show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors, including MIWI, the protein through which pachytene piRNAs function. A-MYB regulation of piRNA pathway proteins and piRNA genes creates a coherent feed-forward loop that ensures the robust accumulation of pachytene piRNAs. This regulatory circuit, which can be detected in rooster testes, likely predates the divergence of birds and mammals. PMID:23523368

  8. Selfish supernumerary chromosome reveals its origin as a mosaic of host genome and organellar sequences.

    Science.gov (United States)

    Martis, Mihaela Maria; Klemme, Sonja; Banaei-Moghaddam, Ali Mohammad; Blattner, Frank R; Macas, Jiří; Schmutzer, Thomas; Scholz, Uwe; Gundlach, Heidrun; Wicker, Thomas; Šimková, Hana; Novák, Petr; Neumann, Pavel; Kubaláková, Marie; Bauer, Eva; Haseneyer, Grit; Fuchs, Jörg; Doležel, Jaroslav; Stein, Nils; Mayer, Klaus F X; Houben, Andreas

    2012-08-14

    Supernumerary B chromosomes are optional additions to the basic set of A chromosomes, and occur in all eukaryotic groups. They differ from the basic complement in morphology, pairing behavior, and inheritance and are not required for normal growth and development. The current view is that B chromosomes are parasitic elements comparable to selfish DNA, like transposons. In contrast to transposons, they are autonomously inherited independent of the host genome and have their own mechanisms of mitotic or meiotic drive. Although B chromosomes were first described a century ago, little is known about their origin and molecular makeup. The widely accepted view is that they are derived from fragments of A chromosomes and/or generated in response to interspecific hybridization. Through next-generation sequencing of sorted A and B chromosomes, we show that B chromosomes of rye are rich in gene-derived sequences, allowing us to trace their origin to fragments of A chromosomes, with the largest parts corresponding to rye chromosomes 3R and 7R. Compared with A chromosomes, B chromosomes were also found to accumulate large amounts of specific repeats and insertions of organellar DNA. The origin of rye B chromosomes occurred an estimated ∼1.1-1.3 Mya, overlapping in time with the onset of the genus Secale (1.7 Mya). We propose a comprehensive model of B chromosome evolution, including its origin by recombination of several A chromosomes followed by capturing of additional A-derived and organellar sequences and amplification of B-specific repeats.

  9. Presence of multiple sites containing polar material in spherical Escherichia coli cells that lack MreB.

    Science.gov (United States)

    Nilsen, Trine; Yan, Arthur W; Gale, Gregory; Goldberg, Marcia B

    2005-09-01

    In rod-shaped bacteria, certain proteins are specifically localized to the cell poles. The nature of the positional information that leads to the proper localization of these proteins is unclear. In a screen for factors required for the localization of the Shigella sp. actin assembly protein IcsA to the bacterial pole, a mutant carrying a transposon insertion in mreB displayed altered targeting of IcsA. The phenotype of cells containing a transposon insertion in mreB was indistinguishable from that of cells containing a nonpolar mutation in mreB or that of wild-type cells treated with the MreB inhibitor A22. In cells lacking MreB, a green fluorescent protein (GFP) fusion to a cytoplasmic derivative of IcsA localized to multiple sites. Secreted full-length native IcsA was present in multiple faint patches on the surfaces of these cells in a pattern similar to that seen for the cytoplasmic IcsA-GFP fusion. EpsM, the polar Vibrio cholerae inner membrane protein, also localized to multiple sites in mreB cells and colocalized with IcsA, indicating that localization to multiple sites is not unique to IcsA. Our results are consistent with the requirement, either direct or indirect, for MreB in the restriction of certain polar material to defined sites within the cell and, in the absence of MreB, with the formation of ectopic sites containing polar material.

  10. Identification of the genes required for the culture of Liberibacter crescens, the closest cultured relative of the uncultured Liberibacter plant pathogens

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    Kin-Kwan eLai

    2016-04-01

    Full Text Available Here Tn5 random transposon mutagenesis was used to identify the essential elements for culturing Liberibacter crescens BT-1 that can serve as antimicrobial targets for the closely related pathogens of citrus, Candidatus Liberibacter asiaticus (Las and tomato and potato, Candidatus Liberibacter solanacearum (Lso. In order to gain insight on the virulence, metabolism, and culturability of the pathogens within the genus Liberibacter, a mini-Tn5 transposon derivative system consisting of a gene specifying resistance to kanamycin, flanked by a 19-base-pair terminal repeat sequence of Tn5, was used for the genome-wide mutagenesis of L. crescens BT-1 and created an insertion mutant library. By analyzing the location of insertions using Sanger and Illumina Mi-Seq sequencing, 314 genes are proposed as essential for the culture of L. crescens BT-1 on BM-7 medium. Of those genes, 76 are not present in the uncultured Liberibacter pathogens and, as a result, suggest molecules necessary for the culturing these pathogens. Those molecules include the aromatic amino acids, several vitamins, histidine, cysteine, lipopolysaccharides, and fatty acids. In addition, the 238 essential genes of L. crescens in common with L. asiaticus are potential targets for the development of therapeutics against the disease.

  11. Target Capture during Mos1 Transposition*

    Science.gov (United States)

    Pflieger, Aude; Jaillet, Jerôme; Petit, Agnès; Augé-Gouillou, Corinne; Renault, Sylvaine

    2014-01-01

    DNA transposition contributes to genomic plasticity. Target capture is a key step in the transposition process, because it contributes to the selection of new insertion sites. Nothing or little is known about how eukaryotic mariner DNA transposons trigger this step. In the case of Mos1, biochemistry and crystallography have deciphered several inverted terminal repeat-transposase complexes that are intermediates during transposition. However, the target capture complex is still unknown. Here, we show that the preintegration complex (i.e., the excised transposon) is the only complex able to capture a target DNA. Mos1 transposase does not support target commitment, which has been proposed to explain Mos1 random genomic integrations within host genomes. We demonstrate that the TA dinucleotide used as the target is crucial both to target recognition and in the chemistry of the strand transfer reaction. Bent DNA molecules are better targets for the capture when the target DNA is nicked two nucleotides apart from the TA. They improve strand transfer when the target DNA contains a mismatch near the TA dinucleotide. PMID:24269942

  12. Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments

    Science.gov (United States)

    Miller, Daniel P.; Hutcherson, Justin A.; Wang, Yan; Nowakowska, Zuzanna M.; Potempa, Jan; Yoder-Himes, Deborah R.; Scott, David A.; Whiteley, Marvin; Lamont, Richard J.

    2017-01-01

    Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis, and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism. PMID:28900609

  13. Catalytic properties of RNA polymerases IV and V: accuracy, nucleotide incorporation and rNTP/dNTP discrimination.

    Science.gov (United States)

    Marasco, Michelle; Li, Weiyi; Lynch, Michael; Pikaard, Craig S

    2017-11-02

    All eukaryotes have three essential nuclear multisubunit RNA polymerases, abbreviated as Pol I, Pol II and Pol III. Plants are remarkable in having two additional multisubunit RNA polymerases, Pol IV and Pol V, which synthesize noncoding RNAs that coordinate RNA-directed DNA methylation for silencing of transposons and a subset of genes. Based on their subunit compositions, Pols IV and V clearly evolved as specialized forms of Pol II, but their catalytic properties remain undefined. Here, we show that Pols IV and V differ from one another, and Pol II, in nucleotide incorporation rate, transcriptional accuracy and the ability to discriminate between ribonucleotides and deoxyribonucleotides. Pol IV transcription is considerably more error-prone than Pols II or V, which may be tolerable in its synthesis of short RNAs that serve as precursors for siRNAs targeting non-identical members of transposon families. By contrast, Pol V exhibits high fidelity transcription, similar to Pol II, suggesting a need for Pol V transcripts to faithfully reflect the DNA sequence of target loci to which siRNA-Argonaute silencing complexes are recruited. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments

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    Daniel P. Miller

    2017-08-01

    Full Text Available Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis, and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq. Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism.

  15. Molecular Analysis of VanA Outbreak of Enterococcus faecium in Two Warsaw Hospitals: The Importance of Mobile Genetic Elements

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    Ewa Wardal

    2014-01-01

    Full Text Available Vancomycin-resistant Enterococcus faecium represents a growing threat in hospital-acquired infections. Two outbreaks of this pathogen from neighboring Warsaw hospitals have been analyzed in this study. Pulsed-field gel electrophoresis (PFGE of SmaI-digested DNA, multilocus VNTR analysis (MLVA, and multilocus sequence typing (MLST revealed a clonal variability of isolates which belonged to three main lineages (17, 18, and 78 of nosocomial E. faecium. All isolates were multidrug resistant and carried several resistance, virulence, and plasmid-specific genes. Almost all isolates shared the same variant of Tn1546 transposon, characterized by the presence of insertion sequence ISEf1 and a point mutation in the vanA gene. In the majority of cases, this transposon was located on 50 kb or 100 kb pRUM-related plasmids, which lacked, however, the axe-txe toxin-antitoxin genes. 100 kb plasmid was easily transferred by conjugation and was found in various clonal backgrounds in both institutions, while 50 kb plasmid was not transferable and occurred solely in MT159/ST78 strains that disseminated clonally in one institution. Although molecular data indicated the spread of VRE between two institutions or a potential common source of this alert pathogen, epidemiological investigations did not reveal the possible route by which outbreak strains disseminated.

  16. A New Class of SINEs with snRNA Gene-Derived Heads.

    Science.gov (United States)

    Kojima, Kenji K

    2015-05-27

    Eukaryotic genomes are colonized by various transposons including short interspersed elements (SINEs). The 5' region (head) of the majority of SINEs is derived from one of the three types of RNA genes--7SL RNA, transfer RNA (tRNA), or 5S ribosomal RNA (rRNA)--and the internal promoter inside the head promotes the transcription of the entire SINEs. Here I report a new group of SINEs whose heads originate from either the U1 or U2 small nuclear RNA gene. These SINEs, named SINEU, are distributed among crocodilians and classified into three families. The structures of the SINEU-1 subfamilies indicate the recurrent addition of a U1- or U2-derived sequence onto the 5' end of SINEU-1 elements. SINEU-1 and SINEU-3 are ancient and shared among alligators, crocodiles, and gharials, while SINEU-2 is absent in the alligator genome. SINEU-2 is the only SINE family that was active after the split of crocodiles and gharials. All SINEU families, especially SINEU-3, are preferentially inserted into a family of Mariner DNA transposon, Mariner-N4_AMi. A group of Tx1 non-long terminal repeat retrotransposons designated Tx1-Mar also show target preference for Mariner-N4_AMi, indicating that SINEU was mobilized by Tx1-Mar. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  17. Characterization of Equine Infectious Anemia Virus Integration in the Horse Genome

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    Qiang Liu

    2015-06-01

    Full Text Available Human immunodeficiency virus (HIV-1 has a unique integration profile in the human genome relative to murine and avian retroviruses. Equine infectious anemia virus (EIAV is another well-studied lentivirus that can also be used as a promising retro-transfection vector, but its integration into its native host has not been characterized. In this study, we mapped 477 integration sites of the EIAV strain EIAVFDDV13 in fetal equine dermal (FED cells during in vitro infection. Published integration sites of EIAV and HIV-1 in the human genome were also analyzed as references. Our results demonstrated that EIAVFDDV13 tended to integrate into genes and AT-rich regions, and it avoided integrating into transcription start sites (TSS, which is consistent with EIAV and HIV-1 integration in the human genome. Notably, the integration of EIAVFDDV13 favored long interspersed elements (LINEs and DNA transposons in the horse genome, whereas the integration of HIV-1 favored short interspersed elements (SINEs in the human genome. The chromosomal environment near LINEs or DNA transposons potentially influences viral transcription and may be related to the unique EIAV latency states in equids. The data on EIAV integration in its natural host will facilitate studies on lentiviral infection and lentivirus-based therapeutic vectors.

  18. Sequence and function of LuxO, a negative regulator of luminescence in Vibrio harveyi.

    Science.gov (United States)

    Bassler, B L; Wright, M; Silverman, M R

    1994-05-01

    Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode a function required for the density-dependent response. Transposon Tn5 insertions in the recombinant clone were isolated, and the mutations were transferred to the genome of V. harveyi for examination of mutant phenotypes. Expression of luminescence in V. harveyi strains with transposon insertions in one locus, luxO, was independent of the density of the culture and was similar in intensity to the maximal level observed in wild-type bacteria. Sequence analysis of luxO revealed one open reading frame that encoded a protein, LuxO, similar in amino acid sequence to the response regulator domain of the family of two-component, signal transduction proteins. The constitutive phenotype of LuxO- mutants indicates that LuxO acts negatively to control expression of luminescence, and relief of repression by LuxO in the wild type could result from interactions with other components in the Lux signalling system.

  19. Legionella dumoffii Tex-KL Mutated in an Operon Homologous to traC-traD is Defective in Epithelial Cell Invasion.

    Science.gov (United States)

    Qin, Tian; Ken-Ichiro, Iida; Ren, Hong Yu; Zhou, Hai Jian; Yoshida, Shin-Ichi

    2016-06-01

    To understand the mechanism of invasion by Legionella dumoffii. The L. dumoffii strain Tex-KL was mutated using the Tn903 derivative, Tn903dIIlacZ. After screening 799 transposon insertion mutants, we isolated one defective mutant. We then constructed the gene-disrupted mutant, KL16, and studied its invasion of and intracellular growth in HeLa and A549 cells, and in A/J mice survival experiments. The structure of traC-traD operon was analyzed by RT-PCR. The transposon insertion was in a gene homologous to Salmonella typhi traC, which is required for the assembly of F pilin into the mature F pilus structure and for conjugal DNA transmission. Results from RT-PCR suggested that the traC-traD region formed an operon. We found that when the traC gene was disrupted, invasion and intracellular growth of L. dumoffii Tex-KL were impaired in human epithelial cells. When mice were infected by intranasal inoculation with a traC deficient mutant, their survival significantly increased when compared to mice infected with the wild-type strain.. Our results indicated that the traC-traD operon is required for the invasion and intracellular growth abilities of L. dumoffii Tex-KL in epithelial cells. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  20. Inhibition of apoptosis by Rv2456c through nuclear factor-κB extends the survival of Mycobacterium tuberculosis

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    Kristen L. Jurcic Smith

    2016-01-01

    Full Text Available Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen with several survival mechanisms aimed at subverting the host immune system. Apoptosis has been shown to be mycobactericidal, to activate CD8+ T cells, and to be modulated by mycobacterial proteins. Since few mycobacterial proteins have so far been directly implicated in the interactions between M. Tuberculosis and host cell apoptosis, we screened M. Tuberculosis H37Rv transposon mutants to identify mutants that fail to inhibit cell death (FID. One of these FID mutants, FID19, had a transposon insertion in Rv2456c and is important for survival in host cells. The lack of the protein resulted in enhanced caspase-3 mediated apoptosis, which is probably due to an inability to activate nuclear factor-κB. Additionally, FID19 infection enhanced polyfunctional CD8+ T cells and induced a higher frequency of interferon-γ secreting immune cells in a murine model. Taken together, our data suggest that Rv2456c is important for the survival of H37Rv by subduing the innate and ultimately adaptive immune responses of its host by preventing apoptosis of the infected cell. Better understanding of the host-mycobacterial interactions may be beneficial to develop novel drug targets and engineer more efficacious vaccine strains against tuberculosis.