A Method for Enhancing the Transfection Efficiency of Minipreps Obtained from Plasmid cDNA Libraries

Kiss-Tóth, Endre; Dower, Steven K.; Sayers, Jon R.

doi:10.1006/abio.2000.4858

Cited by 7 publications

(3 citation statements)

References 5 publications

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“…Earlier models of the T5FEN-DNA interaction threaded single-stranded DNA through the arch. 5,14 However the recent observation that hFEN can catalyse structure-specific cleavage of bifurcated DNA structures that have an oligodeoxynucleotide hybridised to their single-stranded regions and therefore no free 5′ terminus, 20 and that T5FEN can cleave closed circular DNA substrates, 36,38,39 suggests that threading cannot be operational in these substrates. It would therefore appear more likely that 5′-singlestranded DNA passes in front of the helical arch, or is clamped by the helical arch folding over the DNA.…”

Section: Discussionmentioning

confidence: 94%

Comparison of the Catalytic Parameters and Reaction Specificities of a Phage and an Archaeal Flap Endonuclease

Williams

Sengerová

Osborne

et al. 2007

Journal of Molecular Biology

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Flap endonucleases (FENs) catalyse the exonucleolytic hydrolysis of blunt-ended duplex DNA substrates and the endonucleolytic cleavage of 5'-bifurcated nucleic acids at the junction formed between single and double-stranded DNA. The specificity and catalytic parameters of FENs derived from T5 bacteriophage and Archaeoglobus fulgidus were studied with a range of single oligonucleotide DNA substrates. These substrates contained one or more hairpin turns and mimic duplex, 5'-overhanging duplex, pseudo-Y, nicked DNA, and flap structures. The FEN-catalysed reaction properties of nicked DNA and flap structures possessing an extrahelical 3'-nucleotide (nt) were also characterised. The phage enzyme produced multiple reaction products of differing length with all the substrates tested, except when the length of duplex DNA downstream of the reaction site was truncated. Only larger DNAs containing two duplex regions are effective substrates for the archaeal enzyme and undergo reaction at multiple sites when they lack a 3'-extrahelical nucleotide. However, a single product corresponding to reaction 1 nt into the double-stranded region occurred with A. fulgidus FEN when substrates possessed a 3'-extrahelical nt. Steady-state and pre-steady-state catalytic parameters reveal that the phage enzyme is rate-limited by product release with all the substrates tested. Single-turnover maximal rates of reaction are similar with most substrates. In contrast, turnover numbers for T5FEN decrease as the size of the DNA substrate is increased. Comparison of the catalytic parameters of the A. fulgidus FEN employing flap and double-flap substrates indicates that binding interactions with the 3'-extrahelical nucleotide stabilise the ground state FEN-DNA interaction, leading to stimulation of comparative reactions at DNA concentrations below saturation with the single flap substrate. Maximal multiple turnover rates of the archaeal enzyme with flap and double flap substrates are similar. A model is proposed to account for the varying specificities of the two enzymes with regard to cleavage patterns and substrate preferences.

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Section: Discussionmentioning

confidence: 94%

Comparison of the Catalytic Parameters and Reaction Specificities of a Phage and an Archaeal Flap Endonuclease

Williams

Sengerová

Osborne

et al. 2007

Journal of Molecular Biology

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“…The presence of nicked and linear product could affect transfection efficiencies. In order to determine whether the presence of nicked vector affects transfection efficiency, we compared EGFP constructs purified using anion-exchange columns with or without enzymatic digestion of nicked, linear, and ssDNA using T5 exonuclease [ 15 , 27 ], and EGFP bacterial maxiprep. T5 exonuclease treatment followed by anion-exchange column purification results in highly pure closed circular product ( Fig 6A and 6B ), albeit at the cost of reduction in yield.…”

Section: Resultsmentioning

confidence: 99%

Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells

et al. 2016

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Mammalian cells are constantly and unavoidably exposed to DNA damage from endogenous and exogenous sources, frequently to the detriment of genomic integrity and biological function. Cells acquire a large number of chemically diverse lesions per day, and each can have a different genetic fate and biological consequences. However, our knowledge of how and when specific lesions are repaired or how they may compromise the fidelity of DNA replication or transcription and lead to deleterious biological endpoints in mammalian cells is limited. Studying individual lesions requires technically challenging approaches for the targeted introduction of defined lesions into relevant DNA sequences of interest. Here, we present a systematic analysis of factors influencing yield and an improved, efficient and reliable protocol for the production of mammalian expression phagemid vectors containing defined DNA base modifications in any sequence position of either complementary DNA strand. We applied our improved protocol to study the transcriptional mutagenesis-mediated phenotypic consequences of the common oxidative lesion 5-hydroxyuracil, placed in the G12 mutational hotspot of the KRAS oncogene. 5-OHU induced sustained oncogenic signaling in Neil1-/-Neil2-/- mouse cells. The resulting advance in technology will have broad applicability for investigation of single lesion DNA repair, mutagenesis, and DNA damage responses in mammalian cells.

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“…To complement these efforts, we have recently developed a novel screening strategy to enable rapid functional assignment of proteins expressed from cDNA libraries [2][3][4][5]. The rapid progress in recent years has revealed close to the complete map of the euchromatic regions of the human genome with very significant advances being made in mapping the entire human transcriptome as well, a project that is being replicated in many other uni-and multi-cellular organisms.…”

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confidence: 99%

Functional mapping of Toll/interleukin-1 signalling networks by expression cloning

Kiss-Tóth

Wyllie

Holland

et al. 2005

Biochemical Society Transactions

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Multiple cellular proteins have been identified as participating in Toll/interleukin-1 receptor-mediated inflammatory gene expression. The continuing isolation of novel components, based on sequence similarities, protein-protein interactions and protein purification, suggests that many elements of this signalling network remain to be identified. We report here the development of a high-throughput functional screening platform and its application for the identification of components of inflammatory signalling networks. Our results enable us to estimate that 100-150 gene products are involved in controlling the transcription of the human interleukin 8 gene. The approach, which is simple and robust, constitutes a general method for mapping signal transduction systems and for rapid isolation of a large number of signalling components based on the control of pathways leading to regulation of gene expression.

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A Method for Enhancing the Transfection Efficiency of Minipreps Obtained from Plasmid cDNA Libraries

Cited by 7 publications

References 5 publications

Comparison of the Catalytic Parameters and Reaction Specificities of a Phage and an Archaeal Flap Endonuclease

Comparison of the Catalytic Parameters and Reaction Specificities of a Phage and an Archaeal Flap Endonuclease

Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells

Functional mapping of Toll/interleukin-1 signalling networks by expression cloning

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