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Journal ArticleDOI

Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments

01 Oct 2006-Nucleic Acids Research (Oxford University Press)-Vol. 34, Iss: 18
TL;DR: The largely unused uracil-excision molecular cloning technique is advanced by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy.
Abstract: The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy. The original features of the technique, namely simplicity, speed, high efficiency and low cost are thus combined with high fidelity as well as a transparent, simple and flexible vector design. A comprehensive set of vectors has been constructed covering a wide range of different applications and their functionality has been confirmed.

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Citations
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Journal ArticleDOI
TL;DR: It is demonstrated that SLAC1 represents the slow, deactivating, weak voltage-dependent anion channel of guard cells controlled by phosphorylation/dephosphorylation.
Abstract: In response to drought stress the phytohormone ABA (abscisic acid) induces stomatal closure and, therein, activates guard cell anion channels in a calcium-dependent as well as-independent manner. Two key components of the ABA signaling pathway are the protein kinase OST1 (open stomata 1) and the protein phosphatase ABI1 (ABA insensitive 1). The recently identified guard cell anion channel SLAC1 appeared to be the key ion channel in this signaling pathway but remained electrically silent when expressed heterologously. Using split YFP assays, we identified OST1 as an interaction partner of SLAC1 and ABI1. Upon coexpression of SLAC1 with OST1 in Xenopus oocytes, SLAC1-related anion currents appeared similar to those observed in guard cells. Integration of ABI1 into the SLAC1/OST1 complex, however, prevented SLAC1 activation. Our studies demonstrate that SLAC1 represents the slow, deactivating, weak voltage-dependent anion channel of guard cells controlled by phosphorylation/dephosphorylation.

756 citations


Cites methods from "Advancing uracil-excision based clo..."

  • ...The cDNA of SLAC1, OST1, and ABI1 were cloned into plant binary vectors (based on pCAMBIA vectors) as described by Nour-Eldin et al. (2006) (63)....

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Journal ArticleDOI
TL;DR: In this article, the authors review various strategies for small RNA-based gene silencing, and describe in detail the design and application of amiRNAs in many plant species.
Abstract: Comprehensive analysis of gene function requires the detailed examination of mutant alleles In Arabidopsis thaliana, large collections of sequence-indexed insertion and chemical mutants provide potential loss-of-function alleles for most annotated genes However, limitations for phenotypic analysis include gametophytic or early sporophytic lethality, and the ability to recombine mutant alleles in closely linked genes, especially those present as tandem duplications Transgene-mediated gene silencing can overcome some of these shortcomings through tissue-specific, inducible and partial gene inactivation, or simultaneous targeting of several, sequence-related genes In addition, gene silencing is a convenient approach in species or varieties for which exhaustive mutant collections are not yet available Typically, gene function is reduced post-transcriptionally, effected by small RNAs that act in a sequence-specific manner by base pairing to complementary mRNA molecules A recently introduced approach is the use of artificial microRNAs (amiRNAs) Here, we review various strategies for small RNA-based gene silencing, and describe in detail the design and application of amiRNAs in many plant species

659 citations

Journal ArticleDOI
TL;DR: It is argued that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.
Abstract: The 'Standard European Vector Architecture' database (SEVA-DB, http://seva.cnb.csic.es) was conceived as a user-friendly, web-based resource and a material clone repository to assist in the choice of optimal plasmid vectors for de-constructing and re-constructing complex prokaryotic phenotypes. The SEVA-DB adopts simple design concepts that facilitate the swapping of functional modules and the extension of genome engineering options to microorganisms beyond typical laboratory strains. Under the SEVA standard, every DNA portion of the plasmid vectors is minimized, edited for flaws in their sequence and/or functionality, and endowed with physical connectivity through three inter-segment insulators that are flanked by fixed, rare restriction sites. Such a scaffold enables the exchangeability of multiple origins of replication and diverse antibiotic selection markers to shape a frame for their further combination with a large variety of cargo modules that can be used for varied end-applications. The core collection of constructs that are available at the SEVA-DB has been produced as a starting point for the further expansion of the formatted vector platform. We argue that adoption of the SEVA format can become a shortcut to fill the phenomenal gap between the existing power of DNA synthesis and the actual engineering of predictable and efficacious bacteria.

548 citations


Cites background from "Advancing uracil-excision based clo..."

  • ...Finally, one could entertain the assembly of plasmids à la carte by recombining relevant DNA sequences in vitro with suitable enzymatic cocktails (25,26)....

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Journal ArticleDOI
TL;DR: The CPK and OST1 branch of ABA signal transduction in guard cells seem to converge on the level of SLAC1 under the control of the ABI1/ABA-receptor complex.
Abstract: In response to drought stress, the phytohormone abscisic acid (ABA) induces stomatal closure. Thereby the stress hormone activates guard cell anion channels in a calcium-dependent, as well as -independent, manner. Open stomata 1 protein kinase (OST1) and ABI1 protein phosphatase (ABA insensitive 1) represent key components of calcium-independent ABA signaling. Recently, the guard cell anion channel SLAC1 was identified. When expressed heterologously SLAC1 remained electrically silent. Upon coexpression with Ca(2+)-independent OST1, however, SLAC1 anion channels appear activated in an ABI1-dependent manner. Mutants lacking distinct calcium-dependent protein kinases (CPKs) appeared impaired in ABA stimulation of guard cell ion channels, too. To study SLAC1 activation via the calcium-dependent ABA pathway, we studied the SLAC1 response to CPKs in the Xenopus laevis oocyte system. Split YFP-based protein-protein interaction assays, using SLAC1 as the bait, identified guard cell expressed CPK21 and 23 as major interacting partners. Upon coexpression of SLAC1 with CPK21 and 23, anion currents document SLAC1 stimulation by these guard cell protein kinases. Ca(2+)-sensitive activation of SLAC1, however, could be assigned to the CPK21 pathway only because CPK23 turned out to be rather Ca(2+)-insensitive. In line with activation by OST1, CPK activation of the guard cell anion channel was suppressed by ABI1. Thus the CPK and OST1 branch of ABA signal transduction in guard cells seem to converge on the level of SLAC1 under the control of the ABI1/ABA-receptor complex.

493 citations

Journal ArticleDOI
05 Aug 2012-Nature
TL;DR: Identifying and characterize two members of the nitrate/peptide transporter family, GTR1 and GTR2, as high-affinity, proton-dependent glucosinolate-specific transporters has agricultural potential as a means to control allocation of defence compounds in a tissue-specific manner.
Abstract: Two high-affinity proton-dependent transporters of glucosinolates have been identified in Arabidopsis and termed GTR1 and GTR2; these transporters are essential for transporting glucosinolates to seeds, offering a means to control the allocation of defence compounds in a tissue-specific manner, which may have agricultural biotechnology implications. Glucosinolates are important plant defence compounds. They are synthesized in various tissues and then translocated to the seeds, where they accumulate. In this study, Barbara Halkier and colleagues examine the molecular basis of this long-distance transport process. They identify two high-affinity, proton-dependent glucosinolate-specific transporters in Arabidopsis, termed GTR1 and GTR2. These transporters control the loading of glucosinolates from the apoplast into the phloem. The authors' specific and complete elimination of glucosinolates from Arabidopsis seeds, combined with the compounds' retention in vegetative tissues, establishes transport engineering as a potential approach for eliminating anti-nutritional natural products in high-value crops. In plants, transport processes are important for the reallocation of defence compounds to protect tissues of high value1, as demonstrated in the plant model Arabidopsis, in which the major defence compounds, glucosinolates2, are translocated to seeds on maturation3. The molecular basis for long-distance transport of glucosinolates and other defence compounds, however, remains unknown. Here we identify and characterize two members of the nitrate/peptide transporter family, GTR1 and GTR2, as high-affinity, proton-dependent glucosinolate-specific transporters. The gtr1 gtr2 double mutant did not accumulate glucosinolates in seeds and had more than tenfold over-accumulation in source tissues such as leaves and silique walls, indicating that both plasma membrane-localized transporters are essential for long-distance transport of glucosinolates. We propose that GTR1 and GTR2 control the loading of glucosinolates from the apoplasm into the phloem. Identification of the glucosinolate transporters has agricultural potential as a means to control allocation of defence compounds in a tissue-specific manner.

392 citations

References
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Journal ArticleDOI
TL;DR: The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
Abstract: Summary The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA

18,757 citations


"Advancing uracil-excision based clo..." refers methods in this paper

  • ...Arabidopsis thaliana plants were transformed by the floral dip method (7)....

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Journal ArticleDOI
TL;DR: The development of an improved version of YFP named Venus, which contains a novel mutation, F46L, which at 37°C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation and will enable fluorescent labelings that were not possible before.
Abstract: The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2) However, slow maturation is still a big obstacle to the use of GFP variants for visualization These problems are exacerbated when GFP variants are expressed at 37 degrees C and/or targeted to certain organelles Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-) For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired Here we describe the development of an improved version of YFP named "Venus" Venus contains a novel mutation, F46L, which at 37 degrees C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl- We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells Its secretion was readily monitored by measuring release of fluorescence into the medium The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before

2,830 citations


"Advancing uracil-excision based clo..." refers background or methods in this paper

  • ...laevis oocytes T7 For N-terminal fusions to YFP(17)....

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  • ...laevis oocytes T7 For C-terminal fusions to YFP (17)....

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  • ...laevis oocytes T7 For C-terminal fusions to the N-terminal part of YFP for use in BiFC (17,18)....

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  • ...With this approach, we inserted YFP (17), YFP-based bimolecular fluorescence complementation (BiFC) tags (18) and RGSHis6 tags into our existing USER vectors to construct a comprehensive set of C- and N-terminal translational fusion vectors for subcellular localization, protein–protein interaction and protein purification studies, respectively....

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  • ...laevis oocytes T7 For N-terminal fusions to the C-terminal part of YFP for use in BiFC (17,18)....

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Journal ArticleDOI
01 Jan 1985-Nature
TL;DR: The effects of 5′ deletions in a plant viral promoter in tobacco callus as well as in regenerated plants, includ ing different plant tissues, are analysed to allow a more direct assessment of deletion effects.
Abstract: Although promoter regions for many plant nuclear genes have been sequenced, identification of the active promoter sequence has been carried out only for the octopine synthase promoter. That analysis was of callus tissue and made use of an enzyme assay. We have analysed the effects of 5' deletions in a plant viral promoter in tobacco callus as well as in regenerated plants, including different plant tissues. We assayed the RNA transcription product which allows a more direct assessment of deletion effects. The cauliflower mosaic virus (CaMV) 35S promoter provides a model plant nuclear promoter system, as its double-strand DNA genome is transcribed by host nuclear RNA polymerase II from a CaMV minichromosome. Sequences extending to -46 were sufficient for accurate transcription initiation whereas the region between -46 and -105 increased greatly the level of transcription. The 35S promoter showed no tissue-specificity of expression.

1,674 citations

Journal ArticleDOI
TL;DR: Results attest to the general applicability of the BiFC assay for studies of protein interactions.

1,547 citations


"Advancing uracil-excision based clo..." refers background or methods in this paper

  • ...laevis oocytes T7 For C-terminal fusions to the N-terminal part of YFP for use in BiFC (17,18)....

    [...]

  • ...With this approach, we inserted YFP (17), YFP-based bimolecular fluorescence complementation (BiFC) tags (18) and RGSHis6 tags into our existing USER vectors to construct a comprehensive set of C- and N-terminal translational fusion vectors for subcellular localization, protein–protein interaction and protein purification studies, respectively....

    [...]

  • ...laevis oocytes T7 For N-terminal fusions to the C-terminal part of YFP for use in BiFC (17,18)....

    [...]

  • ...N-terminal part of YFP for use in BiFC (17,18)....

    [...]

  • ...C-terminal part of YFP for use in BiFC (17,18)....

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Journal ArticleDOI
TL;DR: A Ti plasmid mutant was constructed in which all the on‐cogenic functions of the T‐DNA have been deleted and replaced by pBR322 and is proposed as an extremely versatile vector for the introduction of any DNA of interest into plant cells.
Abstract: A Ti plasmid mutant was constructed in which all the on-cogenic functions of the T-DNA have been deleted and replaced by pBR322 This Ti plasmid, pGV3850, still mediates efficient transfer and stabilization of its truncated T-DNA into infected plant cells Moreover, integration and expression of this minimal T-DNA in plant cells does not interfere with normal plant cell differentiation A DNA fragment cloned in a pBR vector can be inserted in the pGV3850 T-region upon a single recombination event through the pBR322 region of pGV3850 producing a co-integrate useful for the transformation of plant cells Based upon these properties, pGV3850 is proposed as an extremely versatile vector for the introduction of any DNA of interest into plant cells

800 citations


"Advancing uracil-excision based clo..." refers methods in this paper

  • ...The resulting vector construct was subsequently transformed by electroporation (5) into Agrobacterium tumefaciens strain C58 (6)....

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