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  • Synthetic Biology and Assembly Cloning  (70)
  • Chromatin and Epigenetics  (39)
  • Land Reform  (38)
  • Oxford University Press  (147)
  • 1
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    Supply Response, Marginal Cost, and Soil Erosion Implications of Stover-based Biofuels (2015)
    Oxford University Press
    Details
    Publication Date: 2015-08-11
    Description: Existing economic analysis of corn stover as an energy feedstock has not considered potential changes in land use associated with different stover prices. We estimate the response of corn stover supply density to its price driven by changes in land use and examine its implications for a processing plant's pricing strategy and marginal cost, as well as associated changes in soil erosion. We find that plants will exploit the intensive margin as well as the extensive margin to secure additional amounts of stover. Our results show, counterintuitively, that a market for stover may result in lower soil erosion due to reallocations of land to continuous corn with removal, which, combined with no-till farming, results in lower soil erosion than the baseline without stover removal. Also contrary to expectations, using cover crops with stover removal may result in higher soil erosion due to land use changes within the fuel shed associated with optimal pricing.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q24 - Land, Q42 - Alternative Energy Sources
    Print ISSN: 2040-5790
    Electronic ISSN: 2040-5804
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
    Published by Oxford University Press
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  • 2
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    Post-translational control of genetic circuits using Potyvirus proteases (2016)
    Oxford University Press
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    Publication Date: 2016-07-28
    Description: Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degraded. Three protease:cleavage site pairs from Potyvirus are shown to be orthogonal and active in exposing degrons, releasing inhibitory domains and cleaving polyproteins. This toolbox is applied to the design of genetic circuits as a means to control regulator activity and degradation. First, we demonstrate that a gate can be constructed by constitutively expressing an inactivated repressor and having an input promoter drive the expression of the protease. It is also shown that the proteolytic release of an inhibitory domain can improve the dynamic range of a transcriptional gate (200-fold repression). Next, we design polyproteins containing multiple repressors and show that their cleavage can be used to control multiple outputs. Finally, we demonstrate that the dynamic range of an output can be improved (8-fold to 190-fold) with the addition of a protease-cleaved degron. Thus, controllable proteolysis offers a powerful tool for modulating and expanding the function of synthetic gene circuits.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 3
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    De novo deciphering three-dimensional chromatin interaction and topological domains by wavelet transformation of epigenetic profiles (2016)
    Oxford University Press
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    Publication Date: 2016-06-21
    Description: Defining chromatin interaction frequencies and topological domains is a great challenge for the annotations of genome structures. Although the chromosome conformation capture (3C) and its derivative methods have been developed for exploring the global interactome, they are limited by high experimental complexity and costs. Here we describe a novel computational method, called CITD, for de novo prediction of the chromatin interaction map by integrating histone modification data. We used the public epigenomic data from human fibroblast IMR90 cell and embryonic stem cell (H1) to develop and test CITD, which can not only successfully reconstruct the chromatin interaction frequencies discovered by the Hi-C technology, but also provide additional novel details of chromosomal organizations. We predicted the chromatin interaction frequencies, topological domains and their states (e.g. active or repressive) for 98 additional cell types from Roadmap Epigenomics and ENCODE projects. A total of 131 protein-coding genes located near 78 preserved boundaries among 100 cell types are found to be significantly enriched in functional categories of the nucleosome organization and chromatin assembly. CITD and its predicted results can be used for complementing the topological domains derived from limited Hi-C data and facilitating the understanding of spatial principles underlying the chromosomal organization.
    Keywords: Chromatin and Epigenetics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 4
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    Inducible DamID systems for genomic mapping of chromatin proteins in Drosophila (2016)
    Oxford University Press
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    Publication Date: 2016-07-09
    Description: Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity, it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila . Here, we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress possible toxic effects of Dam. In addition, we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. These new DamID tools will be valuable for the mapping of binding patterns of chromatin proteins in Drosophila tissues and especially in cell lineages.
    Keywords: Chromatin and Epigenetics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 5
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    An efficient strategy for TALEN-mediated genome engineering in Drosophila (2013)
    Oxford University Press
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    Publication Date: 2013-09-26
    Description: In reverse genetics, a gene’s function is elucidated through targeted modifications in the coding region or associated DNA cis -regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila . We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F 1 generation.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 6
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    High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases (2013)
    Oxford University Press
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    Publication Date: 2013-06-08
    Description: Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ~40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 7
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    Cell-free co-production of an orthogonal transfer RNA activates efficient site-specific non-natural amino acid incorporation (2013)
    Oxford University Press
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    Publication Date: 2013-06-08
    Description: We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9–1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p -azido- l -phenylalanine (pAzF) or p -propargyloxy- l -phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50–88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA opt ) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 8
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    In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes (2013)
    Oxford University Press
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    Publication Date: 2013-04-02
    Description: DNA methylation is one of the most important epigenetic alterations involved in the control of gene expression. Bisulfite sequencing of genomic DNA is currently the only method to study DNA methylation patterns at single-nucleotide resolution. Hence, next-generation sequencing of bisulfite-converted DNA is the method of choice to investigate DNA methylation profiles at the genome-wide scale. Nevertheless, whole genome sequencing for analysis of human methylomes is expensive, and a method for targeted gene analysis would provide a good alternative in many cases where the primary interest is restricted to a set of genes. Here, we report the successful use of a custom Agilent SureSelect Target Enrichment system for the hybrid capture of bisulfite-converted DNA. We prepared bisulfite-converted next-generation sequencing libraries, which are enriched for the coding and regulatory regions of 174 ADME genes (i.e. genes involved in the metabolism and distribution of drugs). Sequencing of these libraries on Illumina’s HiSeq2000 revealed that the method allows a reliable quantification of methylation levels of CpG sites in the selected genes, and validation of the method using pyrosequencing and the Illumina 450K methylation BeadChips revealed good concordance.
    Keywords: Chromatin and Epigenetics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 9
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    Highly efficient CRISPR/Cas9-mediated TAR cloning of genes and chromosomal loci from complex genomes in yeast (2015)
    Oxford University Press
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    Publication Date: 2015-05-03
    Description: Transformation-associated recombination (TAR) protocol allowing the selective isolation of full-length genes complete with their distal enhancer regions and entire genomic loci with sizes up to 250 kb from complex genomes in yeast S. cerevisiae has been developed more than a decade ago. However, its wide spread usage has been impeded by a low efficiency (0.5–2%) of chromosomal region capture during yeast transformants which in turn requires a time-consuming screen of hundreds of colonies. Here, we demonstrate that pre-treatment of genomic DNA with CRISPR-Cas9 nucleases to generate double-strand breaks near the targeted genomic region results in a dramatic increase in the fraction of gene-positive colonies (up to 32%). As only a dozen or less yeast transformants need to be screened to obtain a clone with the desired chromosomal region, extensive experience with yeast is no longer required. A TAR-CRISPR protocol may help to create a bank of human genes, each represented by a genomic copy containing its native regulatory elements, that would lead to a significant advance in functional, structural and comparative genomics, in diagnostics, gene replacement, generation of animal models for human diseases and has a potential for gene therapy.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 10
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    A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications (2015)
    Oxford University Press
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    Publication Date: 2015-04-21
    Description: RNA research and therapy relies primarily on synthetic RNAs. We employed recombinant RNA technology toward large-scale production of pre-miRNA agents in bacteria, but found the majority of target RNAs were not or negligibly expressed. We thus developed a novel strategy to achieve consistent high-yield biosynthesis of chimeric RNAs carrying various small RNAs (e.g. miRNAs, siRNAs and RNA aptamers), which was based upon an optimal noncoding RNA scaffold (OnRS) derived from tRNA fusion pre-miR-34a (tRNA/mir-34a). Multi-milligrams of chimeric RNAs (e.g. OnRS/miR-124, OnRS/GFP-siRNA, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 l bacterial culture. Deep sequencing analyses revealed that mature miR-124 and target GFP-siRNA were selectively released from chimeric RNAs in human cells. Consequently, OnRS/miR-124 was active in suppressing miR-124 target gene expression and controlling cellular processes, and OnRS/GFP-siRNA was effective in knocking down GFP mRNA levels and fluorescent intensity in ES-2/GFP cells and GFP -transgenic mice. Furthermore, the OnRS/MGA sensor offered a specific strong fluorescence upon binding MG, which was utilized as label-free substrate to accurately determine serum RNase activities in pancreatic cancer patients. These results demonstrate that OnRS-based bioengineering is a common, robust and versatile strategy to assemble various types of small RNAs for broad applications.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 11
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    Is there a term structure in land lease rates? (2015)
    Oxford University Press
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    Publication Date: 2015-12-29
    Description: This article applies the concept of a term structure to agricultural land rental prices. Based on theoretical considerations, we develop a hedonic pricing model that allows for different shapes of the term structure curve while controlling for other price-relevant characteristics. We apply this model to land lease contracts in Saxony-Anhalt. We find an upward-sloping term structure during the agricultural price boom in 2007 and 2008, where market participants expected increasing rental prices. For the subsequent years, however, we detect a single-humped term structure. Hence, market participants revised their expectations and assumed a decline of land rental prices in the long term.
    Keywords: D44 - Auctions, E43 - Determination of Interest Rates ; Term Structure of Interest Rates, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
    Published by Oxford University Press
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  • 12
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    Nano-mechanical measurements of protein-DNA interactions with a silicon nitride pulley (2016)
    Oxford University Press
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    Publication Date: 2016-01-09
    Description: Proteins adhere to DNA at locations and with strengths that depend on the protein conformation, the underlying DNA sequence and the ionic content of the solution. A facile technique to probe the positions and strengths of protein-DNA binding would aid in understanding these important interactions. Here, we describe a ‘DNA pulley’ for position-resolved nano-mechanical measurements of protein-DNA interactions. A molecule of DNA is tethered by one end to a glass surface, and by the other end to a magnetic bead. The DNA is stretched horizontally by a magnet, and a nanoscale knife made of silicon nitride is manipulated to contact, bend and scan along the DNA. The mechanical profile of the DNA at the contact with the knife is probed via nanometer-precision optical tracking of the magnetic bead. This system enables detection of protein bumps on the DNA and localization of their binding sites. We study theoretically the technical requirements to detect mechanical heterogeneities in the DNA itself.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 13
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    Dealing with the genetic load in bacterial synthetic biology circuits: convergences with the Ohm's law (2016)
    Oxford University Press
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    Publication Date: 2016-01-09
    Description: Synthetic biology seeks to envision living cells as a matter of engineering. However, increasing evidence suggests that the genetic load imposed by the incorporation of synthetic devices in a living organism introduces a sort of unpredictability in the design process. As a result, individual part characterization is not enough to predict the behavior of designed circuits and thus, a costly trial-error process is eventually required. In this work, we provide a new theoretical framework for the predictive treatment of the genetic load. We mathematically and experimentally demonstrate that dependences among genes follow a quantitatively predictable behavior. Our theory predicts the observed reduction of the expression of a given synthetic gene when an extra genetic load is introduced in the circuit. The theory also explains that such dependence qualitatively differs when the extra load is added either by transcriptional or translational modifications. We finally show that the limitation of the cellular resources for gene expression leads to a mathematical formulation that converges to an expression analogous to the Ohm's law for electric circuits. Similitudes and divergences with this law are outlined. Our work provides a suitable framework with predictive character for the design process of complex genetic devices in synthetic biology.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 14
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    Irrigation Decisions for Major West Coast Crops: Water Scarcity and Climatic Determinants (2015)
    Oxford University Press
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    Publication Date: 2015-12-13
    Description: This article uses the 2007 Farm and Ranch Irrigation Survey database developed by the U.S. Department of Agriculture to assess the impact of water scarcity and climate on irrigation decisions for producers of specialty crops, wheat, and forage crops. We estimate an irrigation management model for major crops in the West Coast (California, Oregon, and Washington), which includes a farm-level equation of irrigated share and crop-specific equations of technology adoption and water application rate (orchard/vineyard, vegetable, wheat, alfalfa, hay, and pasture). We find that economic and physical water scarcity, climate, and extreme weather, such as frost, extreme heat, and drought, significantly impact producers’ irrigation decisions. Producers use sprinkler technologies or additional water applications to mitigate risk of crop damage from extreme weather. Water application rates are least responsive to surface water cost or groundwater well depth for producers of orchard/vineyard. Water supply institutions influence producers’ irrigation decisions. Producers who receive water from federal agencies use higher water application rates and are less likely to adopt water-saving irrigation technologies for some crops. Institutional arrangements, including access to distinct water sources (surface or ground) and whether surface water cost is fee based, also affect the responsiveness of water application rates to changes in surface water cost. The analysis provides valuable information about how producers in irrigated agricultural production systems would respond and adapt to water pricing policies and climate change.
    Keywords: Q12 - Micro Analysis of Farm Firms, Farm Households, and Farm Input Markets, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q16 - R&D ; Agricultural Technology ; Agricultural Extension Services, Q18 - Agricultural Policy ; Food Policy, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 15
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    The Economic Costs of Environmental Regulation in U.S. Dairy Farming: A Directional Distance Function Approach (2015)
    Oxford University Press
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    Publication Date: 2015-07-10
    Description: Analyses of the costs of regulating greenhouse gas emissions from dairy production, which could be used to assess the effectiveness of alternative policy measures, is a missing link in the literature. This article addresses this gap by establishing the economic impact associated with a hypothetical greenhouse gas environmental regulatory regime across major dairy producing counties in the United States. In doing so, the article makes three important contributions to the literature. First, it develops a comprehensive pollution index based on Environmental Protection Agency methodologies, which contrasts with previous studies that rely on partial measures based only on surplus nitrogen stemming from the over-application of fertilizer. Second, the article uses a directional output distance function, an approach that has not been employed previously to evaluate polluting technologies in the U.S. dairy sector. Third, the article incorporates a four-way error approach that accounts for unobserved county heterogeneity, time-invariant persistent technical efficiency, time-varying transient technical efficiency, and a random error. The results indicate that regulating greenhouse gas emissions from dairy farming would induce a 5-percentage point increase in average technical efficiency. In addition, the economic costs of implementing this hypothetical regulatory framework exhibit significant spatial variation across counties in the United States.
    Keywords: D22 - Firm Behavior: Empirical Analysis, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q52 - Pollution Control Costs ; Distributional Effects ; Employment Effects
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 16
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    Blue light-mediated transcriptional activation and repression of gene expression in bacteria (2016)
    Oxford University Press
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    Publication Date: 2016-08-20
    Description: Light-regulated modules offer unprecedented new ways to control cellular behavior in precise spatial and temporal resolution. The availability of such tools may dramatically accelerate the progression of synthetic biology applications. Nonetheless, current optogenetic toolbox of prokaryotes has potential issues such as lack of rapid and switchable control, less portable, low dynamic expression and limited parts. To address these shortcomings, we have engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using the blue light dependent DNA-binding protein EL222. We demonstrated that by modulating the dosage of light pulses or intensity we could control the level of gene expression precisely. We show that both light-inducible and repressible system can function in parallel with high spatial precision in a single cell and can be switched stably between ON- and OFF-states by repetitive pulses of blue light. In addition, the light-inducible and repressible expression kinetics were quantitatively analysed using a mathematical model. We further apply the system, for the first time, to optogenetically synchronize two receiver cells performing different logic behaviors over time using blue light as a molecular clock signal. Overall, our modular approach layers a transformative platform for next-generation light-controllable synthetic biology systems in prokaryotes.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 17
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    Drought Index Insurance for the Central Valley Project in California (2016)
    Oxford University Press
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    Publication Date: 2016-08-20
    Description: A multi-year drought has taken a severe toll on the agricultural economy of California’s Central Valley. Index insurance is an instrument with the potential to protect water users from economic losses due to periodic water shortages. An index insurance product based on the Sacramento Index and adapted to the Central Valley Project water supply is proposed. To address the potential for intertemporal adverse selection, three product designs are suggested: (1) "early bird" insurance; (2) variable premium insurance; and (3) variable deductible insurance. The performance of the designs are assessed using loss functions from the Westlands Water District in the San Joaquin Valley.
    Keywords: Q14 - Agricultural Finance, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 2040-5790
    Electronic ISSN: 2040-5804
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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  • 18
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    CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA (2016)
    Oxford University Press
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    Publication Date: 2016-08-20
    Description: Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated ‘CasHRA ( Cas 9-facilitated H omologous R ecombination A ssembly)’ that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo ; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 ( M inimal G enome of Escherichia coli ) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions.
    Keywords: Synthetic Biology and Assembly Cloning
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    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 19
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    Robust, tunable genetic memory from protein sequestration combined with positive feedback (2015)
    Oxford University Press
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    Publication Date: 2015-10-15
    Description: Natural regulatory networks contain many interacting components that allow for fine-tuning of switching and memory properties. Building simple bistable switches, synthetic biologists have learned the design principles of complex natural regulatory networks. However, most switches constructed so far are so simple (e.g. comprising two regulators) that they are functional only within a limited parameter range. Here, we report the construction of robust, tunable bistable switches in Escherichia coli using three heterologous protein regulators (ExsADC) that are sequestered into an inactive complex through a partner swapping mechanism. On the basis of mathematical modeling, we accurately predict and experimentally verify that the hysteretic region can be fine-tuned by controlling the interactions of the ExsADC regulatory cascade using the third member ExsC as a tuning knob. Additionally, we confirm that a dual-positive feedback switch can markedly increase the hysteretic region, compared to its single-positive feedback counterpart. The dual-positive feedback switch displays bistability over a 10 6 -fold range of inducer concentrations, to our knowledge, the largest range reported so far. This work demonstrates the successful interlocking of sequestration-based ultrasensitivity and positive feedback, a design principle that can be applied to the construction of robust, tunable, and predictable genetic programs to achieve increasingly sophisticated biological behaviors.
    Keywords: Synthetic Biology and Assembly Cloning
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  • 20
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    DREAMing: a simple and ultrasensitive method for assessing intratumor epigenetic heterogeneity directly from liquid biopsies (2015)
    Oxford University Press
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    Publication Date: 2015-12-16
    Description: Many cancers comprise heterogeneous populations of cells at primary and metastatic sites throughout the body. The presence or emergence of distinct subclones with drug-resistant genetic and epigenetic phenotypes within these populations can greatly complicate therapeutic intervention. Liquid biopsies of peripheral blood from cancer patients have been suggested as an ideal means of sampling intratumor genetic and epigenetic heterogeneity for diagnostics, monitoring and therapeutic guidance. However, current molecular diagnostic and sequencing methods are not well suited to the routine assessment of epigenetic heterogeneity in difficult samples such as liquid biopsies that contain intrinsically low fractional concentrations of circulating tumor DNA (ctDNA) and rare epigenetic subclonal populations. Here we report an alternative approach, deemed DREAMing (Discrimination of Rare EpiAlleles by Melt), which uses semi-limiting dilution and precise melt curve analysis to distinguish and enumerate individual copies of epiallelic species at single-CpG-site resolution in fractions as low as 0.005%, providing facile and inexpensive ultrasensitive assessment of locus-specific epigenetic heterogeneity directly from liquid biopsies. The technique is demonstrated here for the evaluation of epigenetic heterogeneity at p14 ARF and BRCA1 gene-promoter loci in liquid biopsies obtained from patients in association with non-small cell lung cancer (NSCLC) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), respectively.
    Keywords: Chromatin and Epigenetics
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    Topics: Biology
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  • 21
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    A polymerase engineered for bisulfite sequencing (2015)
    Oxford University Press
    Details
    Publication Date: 2015-12-16
    Description: Bisulfite sequencing is a key methodology in epigenetics. However, the standard workflow of bisulfite sequencing involves heat and strongly basic conditions to convert the intermediary product 5,6-dihydrouridine-6-sulfonate (dhU6S) (generated by reaction of bisulfite with deoxycytidine (dC)) to uracil (dU). These harsh conditions generally lead to sample loss and DNA damage while milder conditions may result in incomplete conversion of intermediates to uracil. Both can lead to poor recovery of bisulfite-treated DNA by the polymerase chain reaction (PCR) as either damaged DNA and/or intermediates of bisulfite treatment are poor substrate for standard DNA polymerases. Here we describe an engineered DNA polymerase (5D4) with an enhanced ability to replicate and PCR amplify bisulfite-treated DNA due to an ability to bypass both DNA lesions and bisulfite intermediates, allowing significantly milder conversion conditions and increased sensitivity in the PCR amplification of bisulfite-treated DNA. Incorporation of the 5D4 DNA polymerase into the bisulfite sequencing workflow thus promises significant sensitivity and efficiency gains.
    Keywords: Chromatin and Epigenetics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Long-range transcriptional interference in E. coli used to construct a dual positive selection system for genetic switches (2016)
    Oxford University Press
    Details
    Publication Date: 2016-06-03
    Description: We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong 70 dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a ‘forward’ gene interferes with the expression of a ‘reverse’ gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
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    Topics: Biology
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    Establishment of a promoter-based chromatin architecture on recently replicated DNA can accommodate variable inter-nucleosome spacing (2016)
    Oxford University Press
    Details
    Publication Date: 2016-09-03
    Description: Nucleosomes, the fundamental subunits of eukaryotic chromatin, are organized with respect to transcriptional start sites. A major challenge to the persistence of this organization is the disassembly of nucleosomes during DNA replication. Here, we use complimentary approaches to map the locations of nucleosomes on recently replicated DNA. We find that nucleosomes are substantially realigned with promoters during the minutes following DNA replication. As a result, the nucleosomal landscape is largely re-established before newly replicated chromosomes are partitioned into daughter cells and can serve as a platform for the re-establishment of gene expression programmes. When the supply of histones is disrupted through mutation of the chaperone Caf1, a promoter-based architecture is generated, but with increased inter-nucleosomal spacing. This indicates that the chromatin remodelling enzymes responsible for spacing nucleosomes are capable of organizing nucleosomes with a range of different linker DNA lengths.
    Keywords: Chromatin and Epigenetics
    Print ISSN: 0305-1048
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    Topics: Biology
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    NEpiC: a network-assisted algorithm for epigenetic studies using mean and variance combined signals (2016)
    Oxford University Press
    Details
    Publication Date: 2016-09-20
    Description: DNA methylation plays an important role in many biological processes. Existing epigenome-wide association studies (EWAS) have successfully identified aberrantly methylated genes in many diseases and disorders with most studies focusing on analysing methylation sites one at a time. Incorporating prior biological information such as biological networks has been proven to be powerful in identifying disease-associated genes in both gene expression studies and genome-wide association studies (GWAS) but has been under studied in EWAS. Although recent studies have noticed that there are differences in methylation variation in different groups, only a few existing methods consider variance signals in DNA methylation studies. Here, we present a network-assisted algorithm, NEpiC, that combines both mean and variance signals in searching for differentially methylated sub-networks using the protein–protein interaction (PPI) network. In simulation studies, we demonstrate the power gain from using both the prior biological information and variance signals compared to using either of the two or neither information. Applications to several DNA methylation datasets from the Cancer Genome Atlas (TCGA) project and DNA methylation data on hepatocellular carcinoma (HCC) from the Columbia University Medical Center (CUMC) suggest that the proposed NEpiC algorithm identifies more cancer-related genes and generates better replication results.
    Keywords: Chromatin and Epigenetics
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    Topics: Biology
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    Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression (2016)
    Oxford University Press
    Details
    Publication Date: 2016-08-20
    Description: To improve the epigenomic analysis of tissues rich in 5-hydroxymethylcytosine (hmC), we developed a novel protocol called TAB-Methyl-SEQ, which allows for single base resolution profiling of both hmC and 5-methylcytosine by targeted next-generation sequencing. TAB-Methyl-SEQ data were extensively validated by a set of five methodologically different protocols. Importantly, these extensive cross-comparisons revealed that protocols based on Tet1-assisted bisulfite conversion provided more precise hmC values than TrueMethyl-based methods. A total of 109 454 CpG sites were analyzed by TAB-Methyl-SEQ for mC and hmC in 188 genes from 20 different adult human livers. We describe three types of variability of hepatic hmC profiles: (i) sample-specific variability at 40.8% of CpG sites analyzed, where the local hmC values correlate to the global hmC content of livers (measured by LC-MS), (ii) gene-specific variability, where hmC levels in the coding regions positively correlate to expression of the respective gene and (iii) site-specific variability, where prominent hmC peaks span only 1 to 3 neighboring CpG sites. Our data suggest that both the gene- and site-specific components of hmC variability might contribute to the epigenetic control of hepatic genes. The protocol described here should be useful for targeted DNA analysis in a variety of applications.
    Keywords: Chromatin and Epigenetics
    Print ISSN: 0305-1048
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    Topics: Biology
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    Who Really Benefits from Agricultural Subsidies? Evidence from Field-level Data (2016)
    Oxford University Press
    Details
    Publication Date: 2016-07-09
    Description: If agricultural subsidies are largely capitalized into farmland values through their effect on rental rates, then expanding support for agriculture may not benefit farmers who rent the land they farm. Existing evidence on the incidence of subsidies on cash rental rates is mixed. Identification is obscured by unobserved or imprecisely measured factors that tend to be correlated with subsidies, especially land quality and time-varying factors like commodity prices and adverse weather events. A problem that has received less attention is the fact that subsidies and land quality on rented land may differ from owned land. Since most farms possess both rented and owned acreage, farm-level measures of subsidies, land values, and rental rates may bias estimated incidence. Using a new, field-level data set that, for the first time, precisely links subsidies to land parcels, we show that this bias is considerable: where farm-level estimates suggest an incidence of 42–49 cents of the marginal subsidy dollar, field-level estimates from the same farms indicate that landlords capture just 20–28 cents. The size of the farm and the duration of the rental arrangement have substantial effects. Incidence falls by 5–15 cents when doubling total operated acres, and the incidence falls by 0.1–0.8 cents with each additional year of the rental arrangement. Low incidence of subsidies on rents combined with the farm-size and duration effects suggest that farmers renting land have monopsony power.
    Keywords: H22 - Incidence, Q14 - Agricultural Finance, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    What Drives the Global "Land Rush"? (2015)
    Oxford University Press
    Details
    Publication Date: 2015-05-21
    Description: We review evidence regarding the size and evolution of the "land rush" in the wake of the 2007–8 boom in agricultural commodity prices, and we study the determinants of foreign land acquisition for large-scale agricultural investment. The use of data on bilateral investment relationships to estimate gravity models of transnational land-intensive investments confirms the central role of agro-ecological potential as a pull factor. However, this finding contrasts the standard literature insofar as the quality of the destination country's business climate is insignificant, and weak tenure security is associated with increased interest for investors to acquire land in the country. Policy implications are discussed.
    Keywords: F21 - International Investment ; Long-Term Capital Movements, O13 - Agriculture ; Natural Resources ; Energy ; Environment ; Other Primary Products, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q34 - Natural Resources and Domestic and International Conflicts
    Print ISSN: 0258-6770
    Electronic ISSN: 1564-698X
    Topics: Economics
    Published by Oxford University Press
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    Irrigated Agricultural Adaptation to Water and Climate Variability: The Economic Value of a Water Portfolio (2015)
    Oxford University Press
    Details
    Publication Date: 2015-04-18
    Description: Increasing aridity, more frequent and intense drought, and greater degrees of water scarcity create unique challenges for agriculture. In response to these challenges, which often manifest themselves as lower and more variable surface water supplies, as well as depleted and degraded ground water supplies, growers tend to seek opportunities to adapt. One option for growers to reduce their exposure to water scarcity and heightened uncertainty is to diversify. Indeed, access to a portfolio of supplies is one way in which water and irrigation districts, as well as individual growers, are responding to the changing landscape of water resource availability. This article evaluates the benefits to irrigated agriculture from having access to multiple sources of water. With farm-level information on 1,900 agricultural parcels across California, we use the hedonic property value method to investigate the extent that growers benefit from having access to multiple sources of water (i.e., a water portfolio). Our results suggest that while lower quality waters, less reliable water, and less water all negatively impact agricultural land values, holding a water portfolio has a positive impact on land values through its role in mitigating the negative aspects of these factors and reducing the sensitivity of agriculture to climate-related factors. From a policy perspective, such results identify a valuable adaptation tool that irrigation districts may consider to help offset the negative impacts of climate change, drought, and population increases on water supply availability and reliability.
    Keywords: Q10 - General, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy, Q50 - General, Q51 - Valuation of Environmental Effects
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Solid-phase cloning for high-throughput assembly of single and multiple DNA parts (2015)
    Oxford University Press
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    Publication Date: 2015-04-21
    Description: We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of 〉60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of 〉150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Identification of protein complexes that bind to histone H3 combinatorial modifications using super-SILAC and weighted correlation network analysis (2015)
    Oxford University Press
    Details
    Publication Date: 2015-02-18
    Description: The large number of chemical modifications that are found on the histone proteins of eukaryotic cells form multiple complex combinations, which can act as recognition signals for reader proteins. We have used peptide capture in conjunction with super-SILAC quantification to carry out an unbiased high-throughput analysis of the composition of protein complexes that bind to histone H3K9/S10 and H3K27/S28 methyl-phospho modifications. The accurate quantification allowed us to perform Weighted correlation network analysis (WGCNA) to obtain a systems-level view of the histone H3 histone tail interactome. The analysis reveals the underlying modularity of the histone reader network with members of nuclear complexes exhibiting very similar binding signatures, which suggests that many proteins bind to histones as part of pre-organized complexes. Our results identify a novel complex that binds to the double H3K9me3/S10ph modification, which includes Atrx, Daxx and members of the FACT complex. The super-SILAC approach allows comparison of binding to multiple peptides with different combinations of modifications and the resolution of the WGCNA analysis is enhanced by maximizing the number of combinations that are compared. This makes it a useful approach for assessing the effects of changes in histone modification combinations on the composition and function of bound complexes.
    Keywords: Chromatin and Epigenetics
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    Topics: Biology
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    Optimization of transcription factor binding map accuracy utilizing knockout-mouse models (2014)
    Oxford University Press
    Details
    Publication Date: 2014-11-28
    Description: Genome-wide assessment of protein–DNA interaction by chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) is a key technology for studying transcription factor (TF) localization and regulation of gene expression. Signal-to-noise-ratio and signal specificity in ChIP-seq studies depend on many variables, including antibody affinity and specificity. Thus far, efforts to improve antibody reagents for ChIP-seq experiments have focused mainly on generating higher quality antibodies. Here we introduce KOIN (knockout implemented normalization) as a novel strategy to increase signal specificity and reduce noise by using TF knockout mice as a critical control for ChIP-seq data experiments. Additionally, KOIN can identify ‘hyper ChIPable regions’ as another source of false-positive signals. As the use of the KOIN algorithm reduces false-positive results and thereby prevents misinterpretation of ChIP-seq data, it should be considered as the gold standard for future ChIP-seq analyses, particularly when developing ChIP-assays with novel antibody reagents.
    Keywords: Chromatin and Epigenetics
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    Post-conversion targeted capture of modified cytosines in mammalian and plant genomes (2015)
    Oxford University Press
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    Publication Date: 2015-07-12
    Description: We present a capture-based approach for bisulfite-converted DNA that allows interrogation of pre-defined genomic locations, allowing quantitative and qualitative assessments of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at CG dinucleotides and in non-CG contexts (CHG, CHH) in mammalian and plant genomes. We show the technique works robustly and reproducibly using as little as 500 ng of starting DNA, with results correlating well with whole genome bisulfite sequencing data, and demonstrate that human DNA can be tested in samples contaminated with microbial DNA. This targeting approach will allow cell type-specific designs to maximize the value of 5mC and 5hmC sequencing.
    Keywords: Chromatin and Epigenetics
    Print ISSN: 0305-1048
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    Topics: Biology
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    Targeting chromatin binding regulation of constitutively active AR variants to overcome prostate cancer resistance to endocrine-based therapies (2015)
    Oxford University Press
    Details
    Publication Date: 2015-07-12
    Description: Androgen receptor (AR) variants (AR-Vs) expressed in prostate cancer (PCa) lack the AR ligand binding domain (LBD) and function as constitutively active transcription factors. AR-V expression in patient tissues or circulating tumor cells is associated with resistance to AR-targeting endocrine therapies and poor outcomes. Here, we investigated the mechanisms governing chromatin binding of AR-Vs with the goal of identifying therapeutic vulnerabilities. By chromatin immunoprecipitation and sequencing (ChIP-seq) and complementary biochemical experiments, we show that AR-Vs display a binding preference for the same canonical high-affinity androgen response elements (AREs) that are preferentially engaged by AR, albeit with lower affinity. Dimerization was an absolute requirement for constitutive AR-V DNA binding and transcriptional activation. Treatment with the bromodomain and extraterminal (BET) inhibitor JQ1 resulted in inhibition of AR-V chromatin binding and impaired AR-V driven PCa cell growth in vitro and in vivo . Importantly, this was associated with a novel JQ1 action of down-regulating AR-V transcript and protein expression. Overall, this study demonstrates that AR-Vs broadly restore AR chromatin binding events that are otherwise suppressed during endocrine therapy, and provides pre-clinical rationale for BET inhibition as a strategy for inhibiting expression and chromatin binding of AR-Vs in PCa.
    Keywords: Chromatin and Epigenetics
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    Enhanced multiplex genome engineering through co-operative oligonucleotide co-selection (2012)
    Oxford University Press
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    Publication Date: 2012-09-27
    Description: Genome-scale engineering of living organisms requires precise and economical methods to efficiently modify many loci within chromosomes. One such example is the directed integration of chemically synthesized single-stranded deoxyribonucleic acid (oligonucleotides) into the chromosome of Escherichia coli during replication. Herein, we present a general co-selection strategy in multiplex genome engineering that yields highly modified cells. We demonstrate that disparate sites throughout the genome can be easily modified simultaneously by leveraging selectable markers within 500 kb of the target sites. We apply this technique to the modification of 80 sites in the E. coli genome.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    Decoupling of agricultural support payments: the impact on land market participation decisions (2012)
    Oxford University Press
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    Publication Date: 2012-08-03
    Description: This paper analyses the impact of the recent decision by the European Union to ‘decouple’ agricultural support payments from agricultural production on Irish farmers' land market decisions. The land market participation decisions of Irish farmers are modelled using a dynamic probit model, while the extent of participation decisions is modelled using a dynamic tobit model. Decoupling does not appear to have significantly altered farmers' land market decisions. One likely explanation for this is the cross-compliance obligation for farmers to maintain land in a state fit for agricultural production in order to receive their full payments.
    Keywords: Q12 - Micro Analysis of Farm Firms, Farm Households, and Farm Input Markets, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Synthesis and cell-free cloning of DNA libraries using programmable microfluidics (2016)
    Oxford University Press
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    Publication Date: 2016-03-01
    Description: Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.
    Keywords: Synthetic Biology and Assembly Cloning
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    A gene network engineering platform for lactic acid bacteria (2016)
    Oxford University Press
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    Publication Date: 2016-03-01
    Description: Recent developments in synthetic biology have positioned lactic acid bacteria (LAB) as a major class of cellular chassis for applications. To achieve the full potential of LAB, one fundamental prerequisite is the capacity for rapid engineering of complex gene networks, such as natural biosynthetic pathways and multicomponent synthetic circuits, into which cellular functions are encoded. Here, we present a synthetic biology platform for rapid construction and optimization of large-scale gene networks in LAB. The platform involves a copy-controlled shuttle for hosting target networks and two associated strategies that enable efficient genetic editing and phenotypic validation. By using a nisin biosynthesis pathway and its variants as examples, we demonstrated multiplex, continuous editing of small DNA parts, such as ribosome-binding sites, as well as efficient manipulation of large building blocks such as genes and operons. To showcase the platform, we applied it to expand the phenotypic diversity of the nisin pathway by quickly generating a library of 63 pathway variants. We further demonstrated its utility by altering the regulatory topology of the nisin pathway for constitutive bacteriocin biosynthesis. This work demonstrates the feasibility of rapid and advanced engineering of gene networks in LAB, fostering their applications in biomedicine and other areas.
    Keywords: Synthetic Biology and Assembly Cloning
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    Genome-wide profiling of nucleosome sensitivity and chromatin accessibility in Drosophila melanogaster (2016)
    Oxford University Press
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    Publication Date: 2016-02-20
    Description: Nucleosomal DNA is thought to be generally inaccessible to DNA-binding factors, such as micrococcal nuclease (MNase). Here, we digest Drosophila chromatin with high and low concentrations of MNase to reveal two distinct nucleosome types: MNase-sensitive and MNase-resistant. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C-containing dinucleotides, whereas MNase-sensitive nucleosomes form on A/T-rich sequences found at transcription start and termination sites, enhancers and DNase I hypersensitive sites. Estimates of nucleosome formation energies indicate that MNase-sensitive nucleosomes tend to be less stable than MNase-resistant ones. Strikingly, a decrease in cell growth temperature of about 10°C makes MNase-sensitive nucleosomes less accessible, suggesting that observed variations in MNase sensitivity are related to either thermal fluctuations of chromatin fibers or the activity of enzymatic machinery. In the vicinity of active genes and DNase I hypersensitive sites nucleosomes are organized into periodic arrays, likely due to ‘phasing’ off potential barriers formed by DNA-bound factors or by nucleosomes anchored to their positions through external interactions. The latter idea is substantiated by our biophysical model of nucleosome positioning and energetics, which predicts that nucleosomes immediately downstream of transcription start sites are anchored and recapitulates nucleosome phasing at active genes significantly better than sequence-dependent models.
    Keywords: Chromatin and Epigenetics
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    ENmix: a novel background correction method for Illumina HumanMethylation450 BeadChip (2016)
    Oxford University Press
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    Publication Date: 2016-02-20
    Description: The Illumina HumanMethylation450 BeadChip is increasingly utilized in epigenome-wide association studies, however, this array-based measurement of DNA methylation is subject to measurement variation. Appropriate data preprocessing to remove background noise is important for detecting the small changes that may be associated with disease. We developed a novel background correction method, ENmix, that uses a mixture of exponential and truncated normal distributions to flexibly model signal intensity and uses a truncated normal distribution to model background noise. Depending on data availability, we employ three approaches to estimate background normal distribution parameters using (i) internal chip negative controls, (ii) out-of-band Infinium I probe intensities or (iii) combined methylated and unmethylated intensities. We evaluate ENmix against other available methods for both reproducibility among duplicate samples and accuracy of methylation measurement among laboratory control samples. ENmix out-performed other background correction methods for both these measures and substantially reduced the probe-design type bias between Infinium I and II probes. In reanalysis of existing EWAS data we show that ENmix can identify additional CpGs, and results in smaller P -value estimates for previously-validated CpGs. We incorporated the method into R package ENmix , which is freely available from Bioconductor website.
    Keywords: Chromatin and Epigenetics
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    Algorithmic co-optimization of genetic constructs and growth conditions: application to 6-ACA, a potential nylon-6 precursor (2015)
    Oxford University Press
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    Publication Date: 2015-12-02
    Description: Optimizing bio-production involves strain and process improvements performed as discrete steps. However, environment impacts genotype and a strain that is optimal under one set of conditions may not be under different conditions. We present a methodology to simultaneously vary genetic and process factors, so that both can be guided by design of experiments (DOE). Advances in DNA assembly and gene insulation facilitate this approach by accelerating multi-gene pathway construction and the statistical interpretation of screening data. This is applied to a 6-aminocaproic acid (6-ACA) pathway in Escherichia coli consisting of six heterologous enzymes. A 32-member fraction factorial library is designed that simultaneously perturbs expression and media composition. This is compared to a 64-member full factorial library just varying expression (0.64 Mb of DNA assembly). Statistical analysis of the screening data from these libraries leads to different predictions as to whether the expression of enzymes needs to increase or decrease. Therefore, if genotype and media were varied separately this would lead to a suboptimal combination. This is applied to the design of a strain and media composition that increases 6-ACA from 9 to 48 mg/l in a single optimization step. This work introduces a generalizable platform to co-optimize genetic and non-genetic factors.
    Keywords: Synthetic Biology and Assembly Cloning
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    Detection of differentially methylated regions from whole-genome bisulfite sequencing data without replicates (2015)
    Oxford University Press
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    Publication Date: 2015-12-02
    Description: DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Recent developments in whole genome bisulfite sequencing (WGBS) technology have enabled genome-wide measurements of DNA methylation at single base pair resolution. Many experiments have been conducted to compare DNA methylation profiles under different biological contexts, with the goal of identifying differentially methylated regions (DMRs). Due to the high cost of WGBS experiments, many studies are still conducted without biological replicates. Methods and tools available for analyzing such data are very limited. We develop a statistical method, DSS-single, for detecting DMRs from WGBS data without replicates. We characterize the count data using a rigorous model that accounts for the spatial correlation of methylation levels, sequence depth and biological variation. We demonstrate that using information from neighboring CG sites, biological variation can be estimated accurately even without replicates. DMR detection is then carried out via a Wald test procedure. Simulations demonstrate that DSS-single has greater sensitivity and accuracy than existing methods, and an analysis of H1 versus IMR90 cell lines suggests that it also yields the most biologically meaningful results. DSS-single is implemented in the Bioconductor package DSS.
    Keywords: Chromatin and Epigenetics
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    Responsiveness of Crop Yield and Acreage to Prices and Climate (2015)
    Oxford University Press
    Details
    Publication Date: 2015-12-13
    Description: We investigate the effect of crop price and climate variables on rainfed corn and soybean yields and acreage in the United States using a large panel dataset for the 1977–2007 period. Instrumental variables are used to control for endogeneity of prices in yield and acreage regressions, while allowing for spatially auto-correlated errors. We find that an increase in corn price has a statistically significant positive impact on corn yield, but the effect of soybean price on soybean yields is not statistically significant. The estimated price elasticities of corn yield and acreage are 0.23 and 0.45, respectively. Of the increase in corn supply caused by an increase in corn price, we find that 33.8% is due to price-induced yield enhancement and 66.2% is due to price-induced acreage expansion. We also find that the impact of climate change on corn production ranges from $-$ 7% to $-$ 41% and on soybean ranges from $-$ 8% to $-$ 45%, depending on the climate change scenarios, time horizon, and global climate models used to predict climate change. We show that the aggregate net impact of omitting price variables is an overestimation of the effect of climate change on corn yield by up to 9% and on soybean yield by up to 15%.
    Keywords: Q11 - Aggregate Supply and Demand Analysis ; Prices, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q54 - Climate ; Natural Disasters ; Global Warming
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Chemical and biological approaches to improve the efficiency of homologous recombination in human cells mediated by artificial restriction DNA cutter (2012)
    Oxford University Press
    Details
    Publication Date: 2012-06-06
    Description: A chemistry-based artificial restriction DNA cutter (ARCUT) was recently prepared from Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acids. This cutter has freely tunable scission-site and site specificity. In this article, homologous recombination (HR) in human cells was promoted by cutting a substrate DNA with ARCUT, and the efficiency of this bioprocess was optimized by various chemical and biological approaches. Of two kinds of terminal structure formed by ARCUT, 3'-overhang termini provided by 1.7-fold higher efficiency than 5'-overhang termini. A longer homology length (e.g. 698 bp) was about 2-fold more favorable than shorter one (e.g. 100 bp). When the cell cycle was synchronized to G2/M phase with nocodazole, the HR was promoted by about 2-fold. Repression of the NHEJ-relevant proteins Ku70 and Ku80 by siRNA increased the efficiency by 2- to 3-fold. It was indicated that appropriate combination of all these chemical and biological approaches should be very effective to promote ARCUT-mediated HR in human cells.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    SLiCE: a novel bacterial cell extract-based DNA cloning method (2012)
    Oxford University Press
    Details
    Publication Date: 2012-04-24
    Description: We describe a novel cloning method termed SLiCE (Seamless L i gation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.
    Keywords: Synthetic Biology and Assembly Cloning
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    Gene targeting to the ROSA26 locus directed by engineered zinc finger nucleases (2012)
    Oxford University Press
    Details
    Publication Date: 2012-04-24
    Description: Targeted gene addition to mammalian genomes is central to biotechnology, basic research and gene therapy. For example, gene targeting to the ROSA26 locus by homologous recombination in embryonic stem cells is commonly used for mouse transgenesis to achieve ubiquitous and persistent transgene expression. However, conventional methods are not readily adaptable to gene targeting in other cell types. The emerging zinc finger nuclease (ZFN) technology facilitates gene targeting in diverse species and cell types, but an optimal strategy for engineering highly active ZFNs is still unclear. We used a modular assembly approach to build ZFNs that target the ROSA26 locus. ZFN activity was dependent on the number of modules in each zinc finger array. The ZFNs were active in a variety of cell types in a time- and dose-dependent manner. The ZFNs directed gene addition to the ROSA26 locus, which enhanced the level of sustained gene expression, the uniformity of gene expression within clonal cell populations and the reproducibility of gene expression between clones. These ZFNs are a promising resource for cell engineering, mouse transgenesis and pre-clinical gene therapy studies. Furthermore, this characterization of the modular assembly method provides general insights into the implementation of the ZFN technology.
    Keywords: Synthetic Biology and Assembly Cloning
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    A transposase strategy for creating libraries of circularly permuted proteins (2012)
    Oxford University Press
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    Publication Date: 2012-05-13
    Description: A simple approach for creating libraries of circularly permuted proteins is described that is called PERMutation Using Transposase Engineering (PERMUTE). In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted. A library of vectors that express different permuted variants of the ORF-encoded protein is created by: (i) using bacteria to select for target vectors that acquire an integrated minitransposon; (ii) excising the ensemble of ORFs that contain an integrated minitransposon from the selected vectors; and (iii) circularizing the ensemble of ORFs containing integrated minitransposons using intramolecular ligation. Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods. In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency (2012)
    Oxford University Press
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    Publication Date: 2012-02-28
    Description: Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli . This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures. Beyond metabolism, we anticipate that DNA scaffolds may be useful in sequestering different types of enzymes for specifying the output of biological signaling pathways or for coordinating other assembly-line processes such as protein folding, degradation and post-translational modifications.
    Keywords: Synthetic Biology and Assembly Cloning
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    Cooperation against theft: A test of incentives for water management In Tunisia (2014)
    Oxford University Press
    Details
    Publication Date: 2014-01-22
    Description: Water theft carried out by manipulating water meters constrains volumetric pricing in semi-arid regions. Cooperative management can reduce theft and improve incentives for efficient water use by inducing peer monitoring. Using a theoretical model, we show that theft is more likely when prices are high, punishments are weak, and cooperatives are large. We also show how cooperative membership and punishment levels are determined endogenously by constraints on monitoring. We test the model on data from Tunisia for the years 2001–2003, relying on instruments that proxy for unobservable monitoring costs. The results confirm that well-designed incentives can reduce theft, and that constraints on monitoring costs affect institutional design.
    Keywords: D82 - Asymmetric and Private Information, Q13 - Agricultural Markets and Marketing ; Cooperatives ; Agribusiness, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q25 - Water
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    A Natural Resource Theory of U.S. Crop Insurance Contract Choice (2014)
    Oxford University Press
    Details
    Publication Date: 2014-01-22
    Description: A large variety of subsidized crop insurance products are available to U.S. crop growers. Distinct and perhaps puzzling patterns in the choices of insurance products and coverage levels can be discerned. Where production conditions are better and yields are less risky then ( a ) higher insurance coverage levels are chosen; and ( b ) revenue insurance is preferred over yield insurance. Also, ( c ) the extent of preference for revenue insurance is stronger in more productive areas. Assuming, as many do, that growers seek to maximize subsidy transfers, point ( a ) can be explained by the interaction between yield technology and natural resource endowments. Points ( b ) and ( c ) can be explained by location in conjunction with the "natural hedge" and a contract design bias in how revenue insurance guarantees are computed. Empirical study of Risk Management Agency data on corn, soybean, and wheat yields, and insurance contract choices lend support to our model inferences.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy, Q24 - Land
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques (2013)
    Oxford University Press
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    Publication Date: 2013-12-07
    Description: The epigenetic modification of 5-hydroxymethylcytosine (5hmC) is receiving great attention due to its potential role in DNA methylation reprogramming and as a cell state identifier. Given this interest, it is important to identify reliable and cost-effective methods for the enrichment of 5hmC marked DNA for downstream analysis. We tested three commonly used affinity-based enrichment techniques; (i) antibody, (ii) chemical capture and (iii) protein affinity enrichment and assessed their ability to accurately and reproducibly report 5hmC profiles in mouse tissues containing high (brain) and lower (liver) levels of 5hmC. The protein-affinity technique is a poor reporter of 5hmC profiles, delivering 5hmC patterns that are incompatible with other methods. Both antibody and chemical capture-based techniques generate highly similar genome-wide patterns for 5hmC, which are independently validated by standard quantitative PCR (qPCR) and glucosyl-sensitive restriction enzyme digestion (gRES-qPCR). Both antibody and chemical capture generated profiles reproducibly link to unique chromatin modification profiles associated with 5hmC. However, there appears to be a slight bias of the antibody to bind to regions of DNA rich in simple repeats. Ultimately, the increased specificity observed with chemical capture-based approaches makes this an attractive method for the analysis of locus-specific or genome-wide patterns of 5hmC.
    Keywords: Chromatin and Epigenetics
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    Topics: Biology
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    Sequence-specific microscopic visualization of DNA methylation status at satellite repeats in individual cell nuclei and chromosomes (2013)
    Oxford University Press
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    Publication Date: 2013-10-19
    Description: Methylation-specific fluorescence in situ hybridization (MeFISH) was developed for microscopic visualization of DNA methylation status at specific repeat sequences in individual cells. MeFISH is based on the differential reactivity of 5-methylcytosine and cytosine in target DNA for interstrand complex formation with osmium and bipyridine-containing nucleic acids (ICON). Cell nuclei and chromosomes hybridized with fluorescence-labeled ICON probes for mouse major and minor satellite repeats were treated with osmium for crosslinking. After denaturation, fluorescent signals were retained specifically at satellite repeats in wild-type, but not in DNA methyltransferase triple-knockout (negative control) mouse embryonic stem cells. Moreover, using MeFISH, we successfully detected hypomethylated satellite repeats in cells from patients with immunodeficiency, centromeric instability and facial anomalies syndrome and 5-hydroxymethylated satellite repeats in male germ cells, the latter of which had been considered to be unmethylated based on anti-5-methylcytosine antibody staining. MeFISH will be suitable for a wide range of applications in epigenetics research and medical diagnosis.
    Keywords: Chromatin and Epigenetics
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    DICE, an efficient system for iterative genomic editing in human pluripotent stem cells (2014)
    Oxford University Press
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    Publication Date: 2014-03-13
    Description: To reveal the full potential of human pluripotent stem cells, new methods for rapid, site-specific genomic engineering are needed. Here, we describe a system for precise genetic modification of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We identified a novel human locus, H11 , located in a safe, intergenic, transcriptionally active region of chromosome 22, as the recipient site, to provide robust, ubiquitous expression of inserted genes. Recipient cell lines were established by site-specific placement of a ‘landing pad’ cassette carrying attP sites for phiC31 and Bxb1 integrases at the H11 locus by spontaneous or TALEN-assisted homologous recombination. Dual integrase cassette exchange (DICE) mediated by phiC31 and Bxb1 integrases was used to insert genes of interest flanked by phiC31 and Bxb1 attB sites at the H11 locus, replacing the landing pad. This system provided complete control over content, direction and copy number of inserted genes, with a specificity of 100%. A series of genes, including mCherry and various combinations of the neural transcription factors LMX1a, FOXA2 and OTX2, were inserted in recipient cell lines derived from H9 ESC, as well as iPSC lines derived from a Parkinson’s disease patient and a normal sibling control. The DICE system offers rapid, efficient and precise gene insertion in ESC and iPSC and is particularly well suited for repeated modifications of the same locus.
    Keywords: Synthetic Biology and Assembly Cloning
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    Improved seamless mutagenesis by recombineering using ccdB for counterselection (2014)
    Oxford University Press
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    Publication Date: 2014-03-13
    Description: Recombineering, which is the use of homologous recombination for DNA engineering in Escherichia coli , usually uses antibiotic selection to identify the intended recombinant. When combined in a second step with counterselection using a small molecule toxin, seamless products can be obtained. Here, we report the advantages of a genetic strategy using CcdB as the counterselectable agent. Expression of CcdB is toxic to E. coli in the absence of the CcdA antidote so counterselection is initiated by the removal of CcdA expression. CcdB counterselection is robust and does not require titrations or experiment-to-experiment optimization. Because counterselection strategies necessarily differ according to the copy number of the target, we describe two variations. For multi-copy targets, we use two E. coli hosts so that counterselection is exerted by the transformation step that is needed to separate the recombined and unrecombined plasmids. For single copy targets, we put the ccdA gene onto the temperature-sensitive pSC101 Red expression plasmid so that counterselection is exerted by the standard temperature shift to remove the expression plasmid. To reduce unwanted intramolecular recombination, we also combined CcdB counterselection with Redα omission. These options improve the use of counterselection in recombineering with BACs, plasmids and the E. coli chromosome.
    Keywords: Synthetic Biology and Assembly Cloning
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    A Bayesian hierarchical model to detect differentially methylated loci from single nucleotide resolution sequencing data (2014)
    Oxford University Press
    Details
    Publication Date: 2014-05-01
    Description: DNA methylation is an important epigenetic modification that has essential roles in cellular processes including gene regulation, development and disease and is widely dysregulated in most types of cancer. Recent advances in sequencing technology have enabled the measurement of DNA methylation at single nucleotide resolution through methods such as whole-genome bisulfite sequencing and reduced representation bisulfite sequencing. In DNA methylation studies, a key task is to identify differences under distinct biological contexts, for example, between tumor and normal tissue. A challenge in sequencing studies is that the number of biological replicates is often limited by the costs of sequencing. The small number of replicates leads to unstable variance estimation, which can reduce accuracy to detect differentially methylated loci (DML). Here we propose a novel statistical method to detect DML when comparing two treatment groups. The sequencing counts are described by a lognormal-beta-binomial hierarchical model, which provides a basis for information sharing across different CpG sites. A Wald test is developed for hypothesis testing at each CpG site. Simulation results show that the proposed method yields improved DML detection compared to existing methods, particularly when the number of replicates is low. The proposed method is implemented in the Bioconductor package DSS.
    Keywords: Chromatin and Epigenetics
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    Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli (2013)
    Oxford University Press
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    Publication Date: 2013-12-07
    Description: The two-step process of selection and counter-selection is a standard way to enable genetic modification and engineering of bacterial genomes using homologous recombination methods. The tetA and sacB genes are contained in a DNA cassette and confer a novel dual counter-selection system. Expression of tetA confers bacterial resistance to tetracycline (Tc R ) and also causes sensitivity to the lipophillic chelator fusaric acid; sacB causes sensitivity to sucrose. These two genes are introduced as a joint DNA cassette into Escherichia coli by selection for Tc R . A medium containing both fusaric acid and sucrose has been developed, in which, coexpression of tetA-sacB is orders of magnitude more sensitive as a counter-selection agent than either gene alone. In conjunction with the homologous recombination methods of recombineering and P1 transduction, this powerful system has been used to select changes in the bacterial genome that cannot be directly detected by other counter-selection systems.
    Keywords: Synthetic Biology and Assembly Cloning
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    High-throughput, cost-effective verification of structural DNA assembly (2014)
    Oxford University Press
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    Publication Date: 2014-02-28
    Description: DNA ‘assembly’ from ‘building blocks’ remains a cornerstone in synthetic biology, whether it be for gene synthesis (~1 kb), pathway engineering (~10 kb) or synthetic genomes (〉100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at 〈$1 per sample.
    Keywords: Synthetic Biology and Assembly Cloning
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    Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination (2014)
    Oxford University Press
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    Publication Date: 2014-02-28
    Description: Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by C31 integrase. Using six orthogonal attP / attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. C31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.
    Keywords: Synthetic Biology and Assembly Cloning
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    Targeting and tracing of specific DNA sequences with dTALEs in living cells (2014)
    Oxford University Press
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    Publication Date: 2014-04-03
    Description: Epigenetic regulation of gene expression involves, besides DNA and histone modifications, the relative positioning of DNA sequences within the nucleus. To trace specific DNA sequences in living cells, we used programmable sequence-specific DNA binding of designer transcription activator-like effectors (dTALEs). We designed a recombinant dTALE (msTALE) with variable repeat domains to specifically bind a 19-bp target sequence of major satellite DNA. The msTALE was fused with green fluorescent protein (GFP) and stably expressed in mouse embryonic stem cells. Hybridization with a major satellite probe (3D-fluorescent in situ hybridization) and co-staining for known cellular structures confirmed in vivo binding of the GFP-msTALE to major satellite DNA present at nuclear chromocenters. Dual tracing of major satellite DNA and the replication machinery throughout S-phase showed co-localization during mid to late S-phase, directly demonstrating the late replication timing of major satellite DNA. Fluorescence bleaching experiments indicated a relatively stable but still dynamic binding, with mean residence times in the range of minutes. Fluorescently labeled dTALEs open new perspectives to target and trace DNA sequences and to monitor dynamic changes in subnuclear positioning as well as interactions with functional nuclear structures during cell cycle progression and cellular differentiation.
    Keywords: Chromatin and Epigenetics
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    Comparison and quantitative verification of mapping algorithms for whole-genome bisulfite sequencing (2014)
    Oxford University Press
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    Publication Date: 2014-04-03
    Description: Coupling bisulfite conversion with next-generation sequencing (Bisulfite-seq) enables genome-wide measurement of DNA methylation, but poses unique challenges for mapping. However, despite a proliferation of Bisulfite-seq mapping tools, no systematic comparison of their genomic coverage and quantitative accuracy has been reported. We sequenced bisulfite-converted DNA from two tissues from each of two healthy human adults and systematically compared five widely used Bisulfite-seq mapping algorithms: Bismark, BSMAP, Pash, BatMeth and BS Seeker. We evaluated their computational speed and genomic coverage and verified their percentage methylation estimates. With the exception of BatMeth, all mappers covered 〉70% of CpG sites genome-wide and yielded highly concordant estimates of percentage methylation ( r 2 ≥ 0.95). Fourfold variation in mapping time was found between BSMAP (fastest) and Pash (slowest). In each library, 8–12% of genomic regions covered by Bismark and Pash were not covered by BSMAP. An experiment using simulated reads confirmed that Pash has an exceptional ability to uniquely map reads in genomic regions of structural variation. Independent verification by bisulfite pyrosequencing generally confirmed the percentage methylation estimates by the mappers. Of these algorithms, Bismark provides an attractive combination of processing speed, genomic coverage and quantitative accuracy, whereas Pash offers considerably higher genomic coverage.
    Keywords: Chromatin and Epigenetics
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    Synthetic biology tools for programming gene expression without nutritional perturbations in Saccharomyces cerevisiae (2014)
    Oxford University Press
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    Publication Date: 2014-04-03
    Description: A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of β-estradiol, so long as cells express the artificial transcription factor, Z 3 EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z 3 EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input–output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z 3 EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications.
    Keywords: Synthetic Biology and Assembly Cloning
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    Capitalized Costs of Habitat Conservation Easements (2014)
    Oxford University Press
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    Publication Date: 2014-04-05
    Description: Perpetual conservation easements permanently remove the option to convert existing habitat to more intensive agricultural production. If existing habitat is at threat of conversion, removing the option to convert will reduce land values. In this article, we estimate the land value discount resulting from perpetual habitat conservation easements by using propensity score matching. We find that on the average eased parcel, land values fall by approximately $86 per acre for every acre of eased habitat. On average, our results suggest that landowners have been adequately compensated and conservation agencies have successfully secured habitat at risk of conversion.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q24 - Land, Q57 - Ecological Economics: Ecosystem Services ; Biodiversity Conservation ; Bioeconomics
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Agri-environmental Policy and Urban Development Patterns: A General Equilibrium Analysis (2014)
    Oxford University Press
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    Publication Date: 2014-04-05
    Description: This paper investigates the spatial effects that the provision of environmental public goods have on residential location choices in a suburban context. Specifically, a spatial general equilibrium framework is developed to analyze the consequences of adopting an agri-environmental policy promoting the provision of positive farming externalities. We use a static monocentric model of an open city where agricultural bid-rents and agricultural amenities vary endogenously in space, and where the positive externalities associated with agricultural production are valued by households. Consistent with empirical evidence of the potential side effects that conservation policies may have in terms of urbanization patterns and land price changes, we show that under certain conditions implementing an agri-environmental policy may promote additional suburban development. Moreover, we demonstrate that the emergence of disconnected suburban areas may be significantly influenced by the location of land regulated by an agri-environmental policy. Finally, we discuss distributional aspects and show that while introducing an agri-environmental policy has a negative impact on most residential land value, it can have positive effects on farmland and residential land located within the regulated areas, suggesting the non-neutrality of such policies regarding the agents’ assets.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy, R13 - General Equilibrium and Welfare Economic Analysis of Regional Economies, R14 - Land Use Patterns, R21 - Housing Demand
    Print ISSN: 0002-9092
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    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Agricultural Rodent Control Using Barn Owls: Is It Profitable? (2014)
    Oxford University Press
    Details
    Publication Date: 2014-04-05
    Description: We develop a model to evaluate the profitability of controlling rodent damage by placing barn owl nesting boxes in agricultural areas. The model incorporates the spatial patterns of barn owl predation pressure on rodents, and the impact of this predation pressure on nesting choices and agricultural output. We apply the model to data collected in Israel and find the installation of nesting boxes profitable. While this finding indicates that economic policy instruments to enhance the adoption of this biological control method are redundant, it does support stricter regulations on rodent control using rodenticides.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy, Q57 - Ecological Economics: Ecosystem Services ; Biodiversity Conservation ; Bioeconomics
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
    Published by Oxford University Press
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    An exogenous chloroplast genome for complex sequence manipulation in algae (2012)
    Oxford University Press
    Details
    Publication Date: 2012-03-29
    Description: We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii . Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus . Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    How do agricultural policies influence farm size inequality? The example of France (2011)
    Oxford University Press
    Details
    Publication Date: 2011-12-27
    Description: We investigate farm size inequality in France using agricultural censuses and farm structure surveys at the NUTS3 level (‘départements’) during the period 1970–2007. Using calculated Gini coefficients, we show that farm size inequality has not systematically increased in France. An econometric analysis of the determinants of farm size inequality reveals that policy measures significantly affected farm size inequality, with most of the measures considered decreasing it. Empirical results suggest that the main contributor was the activity of the SAFER (Société d'Aménagement Foncier et d'Etablissement Rural), a specific feature of the French farm structural policy aimed at regulating rural land management. Besides, this research highlights the great complexity of the dynamics underlying the evolution of farm size distribution.
    Keywords: D30 - General, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Optimisation of implementation policies of environmental quota trade (2011)
    Oxford University Press
    Details
    Publication Date: 2011-12-27
    Description: This paper analyses implementation policies of environmental quota trade, with the Flemish nutrient production rights as an example. Implementation policies concern the transaction quantity, quota reduction and prevention of speculation. They are analysed with a static and a dynamic multi-agent quota trade model. The static model with discrete non-auctioned quota trade shows that the obligation for quota sellers to entirely stop their production stimulates structural change. The dynamic model version indicates that a flat rate reduction on traded quota and measures taken to prevent speculation combined with too low penalties for overuse stimulate the total production.
    Keywords: Q12 - Micro Analysis of Farm Firms, Farm Households, and Farm Input Markets, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    A versatile element for gene addition in bacterial chromosomes (2012)
    Oxford University Press
    Details
    Publication Date: 2012-02-17
    Description: The increasing interest in genetic manipulation of bacterial host metabolic pathways for protein or small molecule production has led to a need to add new genes to a chromosome quickly and easily without leaving behind a selectable marker. The present report describes a vector and four-day procedure that enable site-specific chromosomal insertion of cloned genes in a context insulated from external transcription, usable once in a construction series. The use of rhamnose-inducible transcription from rhaBp allows regulation of the inserted genes independently of the commonly used IPTG and arabinose strategies. Using lacZ as a reporter, we first show that expression from the rhamnose promoter is tightly regulatable, exhibiting very low leakage of background expression compared with background, and moderate rhamnose-induced expression compared with IPTG-induced expression from lacp . Second, the expression of a DNA methyltransferase was used to show that rhamnose regulation yielded on-off expression of this enzyme, such that a resident high-copy plasmid was either fully sensitive or fully resistant to isoschizomer restriction enzyme cleavage. In both cases, growth medium manipulation allows intermediate levels of expression. The vehicle can also be adapted as an ORF-cloning vector.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Error correction of microchip synthesized genes using Surveyor nuclease (2012)
    Oxford University Press
    Details
    Publication Date: 2012-02-17
    Description: The development of economical and high-throughput gene synthesis technology has been hampered by the high occurrence of errors in the synthesized products, which requires expensive labor and time to correct. Here, we describe an error correction reaction (ECR), which employs Surveyor, a mismatch-specific DNA endonuclease, to remove errors from synthetic genes. In ECR reactions, errors are revealed as mismatches by re-annealing of the synthetic gene products. Mismatches are recognized and excised by a combination of mismatch-specific endonuclease and 3'-〉5' exonuclease activities in the reaction mixture. Finally, overlap extension polymerase chain reaction (OE-PCR) re-assembles the resulting fragments into intact genes. The process can be iterated for increased fidelity. With two iterations, we were able to reduce errors in synthetic genes by 〉16-fold, yielding a final error rate of ~1 in 8700 bp.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    The bone-specific Runx2-P1 promoter displays conserved three-dimensional chromatin structure with the syntenic Supt3h promoter (2014)
    Oxford University Press
    Details
    Publication Date: 2014-09-17
    Description: Three-dimensional organization of chromatin is fundamental for transcriptional regulation. Tissue-specific transcriptional programs are orchestrated by transcription factors and epigenetic regulators. The RUNX2 transcription factor is required for differentiation of precursor cells into mature osteoblasts. Although organization and control of the bone-specific Runx2-P1 promoter have been studied extensively, long-range regulation has not been explored. In this study, we investigated higher-order organization of the Runx2-P1 promoter during osteoblast differentiation. Mining the ENCODE database revealed interactions between Runx2-P1 and  Supt3h promoters in several non-mesenchymal human cell lines. Supt3h is a ubiquitously expressed gene located within the first intron of Runx2 . These two genes show shared synteny across species from humans to sponges. Chromosome conformation capture analysis in the murine pre-osteoblastic MC3T3-E1 cell line revealed increased contact frequency between Runx2-P1 and Supt3h promoters during differentiation. This increase was accompanied by enhanced DNaseI hypersensitivity along with RUNX2 and CTCF binding at the Supt3h promoter. Furthermore, interplasmid-3C and luciferase reporter assays showed that the Supt3h promoter can modulate Runx2-P1 activity via direct association. Taken together, our data demonstrate physical proximity between Runx2-P1 and Supt3h promoters, consistent with their syntenic nature. Importantly, we identify the Supt3h promoter as a potential regulator of the bone-specific Runx2-P1 promoter .
    Keywords: Chromatin and Epigenetics
    Print ISSN: 0305-1048
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    Topics: Biology
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    Inducible, tightly regulated and growth condition-independent transcription factor in Saccharomyces cerevisiae (2014)
    Oxford University Press
    Details
    Publication Date: 2014-09-27
    Description: The precise control of gene expression is essential in basic biological research as well as in biotechnological applications. Most regulated systems available in yeast enable only the overexpression of the target gene, excluding the possibility of intermediate or weak expression. Moreover, these systems are frequently toxic or depend on growth conditions. We constructed a heterologous transcription factor that overcomes these limitations. Our system is a fusion of the bacterial LexA DNA-binding protein, the human estrogen receptor (ER) and an activation domain (AD). The activity of this chimera, called LexA-ER-AD, is tightly regulated by the hormone β-estradiol. The selection of the AD proved to be crucial to avoid toxic effects and to define the range of activity that can be precisely tuned with β-estradiol. As our system is based on a heterologous DNA-binding domain, induction in different metabolic contexts is possible. Additionally, by controlling the number of LexA-binding sites in the target promoter, one can scale the expression levels up or down. Overall, our LexA-ER-AD system is a valuable tool to precisely control gene expression in different experimental contexts without toxic side effects.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Broad-host-range vector system for synthetic biology and biotechnology in cyanobacteria (2014)
    Oxford University Press
    Details
    Publication Date: 2014-09-27
    Description: Inspired by the developments of synthetic biology and the need for improved genetic tools to exploit cyanobacteria for the production of renewable bioproducts, we developed a versatile platform for the construction of broad-host-range vector systems. This platform includes the following features: (i) an efficient assembly strategy in which modules released from 3 to 4 donor plasmids or produced by polymerase chain reaction are assembled by isothermal assembly guided by short GC-rich overlap sequences. (ii) A growing library of molecular devices categorized in three major groups: (a) replication and chromosomal integration; (b) antibiotic resistance; (c) functional modules. These modules can be assembled in different combinations to construct a variety of autonomously replicating plasmids and suicide plasmids for gene knockout and knockin. (iii) A web service, the CYANO-VECTOR assembly portal, which was built to organize the various modules, facilitate the in silico construction of plasmids, and encourage the use of this system. This work also resulted in the construction of an improved broad-host-range replicon derived from RSF1010, which replicates in several phylogenetically distinct strains including a new experimental model strain Synechocystis sp. WHSyn, and the characterization of nine antibiotic cassettes, four reporter genes, four promoters, and a ribozyme-based insulator in several diverse cyanobacterial strains.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Intensive and extensive margin adjustments to water scarcity in France's Cereal Belt (2014)
    Oxford University Press
    Details
    Publication Date: 2014-11-11
    Description: Efficient water management in agriculture is becoming critical due to increasing environmental constraints and global food and bio-energy demands. Farmers may respond to increased water scarcity along three main adjustment margins: a move towards rain-fed agriculture (super-extensive margin) or towards less water-intensive crops (extensive margin), and a reduction in water intensity for irrigated crops (intensive margin). Using a positive mathematical programming model of regional supply calibrated to economic and agronomic information, we decompose the total effect of reduced water availability on these adjustment margins in Beauce, a productive cereal region that relies on a groundwater resource to meet its irrigation needs. For realistic water scarcity scenarios, 57 per cent of the total response is attributable to super-extensive margin adjustments. The extensive margin represents 28 per cent of the total response, while the intensive margin accounts for 15 per cent. Crop-level analysis reveals more subtle adaptation patterns.
    Keywords: Q11 - Aggregate Supply and Demand Analysis ; Prices, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q25 - Water
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Encouraging Reductions in Nonpoint Source Pollution through Point-nonpoint Trading: The Roles of Baseline Choice and Practice Subsidies (2014)
    Oxford University Press
    Details
    Publication Date: 2014-09-06
    Description: Water quality regulations in the United States apply almost exclusively to point sources. In impaired watersheds where both point and nonpoint sources contribute to pollution, the U.S. Environmental Protection Agency (EPA) is encouraging the use of point-nonpoint trading to reduce the cost of point sources to meet their permit requirement, and to encourage nonpoint sources to voluntarily contribute more towards meeting overall water quality goals. The EPA guidance encourages trading programs to set a nonpoint source eligibility baseline that extracts some "extra" abatement from nonpoint sources. Research has shown that setting an eligibility baseline that is substantially more stringent than current management could discourage nonpoint source participation and significantly hinder trading. In this paper we examine how choosing the eligibility baseline for agricultural sources affects the efficiency goal of trading (reducing costs to point sources), as well as how it affects the EPA goal of encouraging nonpoint abatement. Using data from the Chesapeake Bay Watershed we find that eligibility baselines set to encourage additional nonpoint source abatement reduce the supply of credits in a market; the more stringent the baseline, the fewer the trades and the smaller the overall abatement from nonpoint sources. A subsidy to farmers for reducing the cost of meeting a baseline encourages greater nonpoint source abatement, but may not benefit the trading market.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q20 - General, Q58 - Government Policy
    Print ISSN: 2040-5790
    Electronic ISSN: 2040-5804
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    A platform for rapid prototyping of synthetic gene networks in mammalian cells (2014)
    Oxford University Press
    Details
    Publication Date: 2014-11-28
    Description: Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
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    Topics: Biology
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    Individual and Cooperative Management of Invasive Species in Human-mediated Landscapes (2014)
    Oxford University Press
    Details
    Publication Date: 2014-12-13
    Description: As a bioinvasion spreads across a landscape from its point of introduction, damages rise roughly with the square of the distance from the original invasion. It is thus generally beneficial, at the landscape scale, to apply eradication or containment controls early if not immediately upon discovery. However, an individual property owner only has incentives to consider the costs and benefits of control on his/her own property rather than potential landscape-scale damages. Bioinvasions will therefore generally be under-controlled in a landscape of independent owners operating under a laissez-faire system. A mechanism is thus needed to induce early cooperative contributions to control costs from beneficiaries who would, without them, be invaded later. We develop a spatially-explicit, integrated model of invasion spread and human behavior to examine how different degrees of spatial cooperation affect patterns of invasion spread and the total costs and damages imposed. We compare individual laissez-faire, cooperative control by adjacent neighbors, and cooperative control by groups including more distant but nearby neighbors. As expected, private laissez-faire control decisions tend to under-control the invasion relative to socially optimal control under most circumstances. But a reasonably high fraction of first best payoffs can be achieved with only a modest geographical reach of cooperation. We also find that less extensive cooperation is needed to control invasions whose costs and damages otherwise lead to the largest externalities (circumstances with costs that are relatively low compared with damages). This suggests that even small amounts of cooperation to control bioinvasions can provide large social benefits.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q24 - Land, Q57 - Ecological Economics: Ecosystem Services ; Biodiversity Conservation ; Bioeconomics
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Farmland Protection and Agricultural Land Values at the Urban-Rural Fringe: British Columbia's Agricultural Land Reserve (2014)
    Oxford University Press
    Details
    Publication Date: 2014-12-13
    Description: Farmland conservation policies typically use zoning and differentiated taxes to prevent urban development of farmland, but little is known about the effectiveness of these policies. This study adds to current knowledge by examining the impact of British Columbia's Agricultural Land Reserve (ALR), established in 1973, which severely restricts subdivision and nonagricultural uses for more than 4.7 million hectares of farmland. To determine the extent to which the ALR preserves farmland by reducing or removing the development option, a multilevel hedonic pricing model is used to estimate the impact of land use, geographic, and zoning characteristics on farmland value near the capital city of Victoria on Vancouver Island. Using sales data from 1974 through 2008, the model demonstrates a changing ALR impact over time that varies considerably by improved and unimproved land types. In 2008, landowners paid 19% less for the typical improved farmland parcel within the ALR versus that outside it. This suggests that would-be developers expect permanency in the zoning law, and prefer non-ALR zoned land. However, ALR land that is unimproved has a premium of 55%, suggesting that this land is more valuable for agriculture than for development. Farmland located closer to the city or the commuting highway commands a premium if it has a residence on it, with a residence also explaining why smaller agricultural properties sell at higher prices. However, it appears that zoning by itself is insufficient to protect farmland; other policies likely need to be implemented in conjunction with zoning to protect agricultural land.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q28 - Government Policy, R14 - Land Use Patterns
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Capitalized Amenity Value of Native Vegetation in a Multifunctional Rural Landscape (2014)
    Oxford University Press
    Details
    Publication Date: 2014-12-13
    Description: In many parts of the world, natural vegetation has been cleared to allow agricultural production. To ensure a long-term flow of ecosystem services without compromising agricultural activities, restoring the environment requires a balance between public and private benefits and costs. Information about private benefits generated by environmental assets can be utilized to identify conservation opportunities on private lands, evaluate environmental projects, and design effective policy instruments. We use a spatio-temporal hedonic model to estimate the private benefits of native vegetation on rural properties in the state of Victoria, Australia. Specifically, we estimate the marginal value of native vegetation on private land and examine how it varies with the extent of vegetation on a property and across a range of property types and sizes. Private benefits of native vegetation are greater per unit area on small and medium-sized properties and smaller on large production-oriented farms. Native vegetation exhibits diminishing marginal benefits as its proportion of a property increases. The current extent of native vegetation cover is lower than the extent that would maximize the amenity value to many landowners. There is scope for improved targeting of investment in the study region by incorporating private benefits of environmental projects into environmental planning processes. Landowners with high marginal private benefits from revegetation would be more willing to participate in a revegetation program. Targeting these landowners would likely provide higher value for money because such projects could be implemented at lower public cost.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q57 - Ecological Economics: Ecosystem Services ; Biodiversity Conservation ; Bioeconomics
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Customized optimization of metabolic pathways by combinatorial transcriptional engineering (2012)
    Oxford University Press
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    Publication Date: 2012-10-10
    Description: A major challenge in metabolic engineering and synthetic biology is to balance the flux of an engineered heterologous metabolic pathway to achieve high yield and productivity in a target organism. Here, we report a simple, efficient and programmable approach named ‘customized optimization of metabolic pathways by combinatorial transcriptional engineering (COMPACTER)’ for rapid tuning of gene expression in a heterologous pathway under distinct metabolic backgrounds. Specifically, a library of mutant pathways is created by de novo assembly of promoter mutants of varying strengths for each pathway gene in a target organism followed by high-throughput screening/selection. To demonstrate this approach, a single round of COMPACTER was used to generate both a xylose utilizing pathway with near-highest efficiency and a cellobiose utilizing pathway with highest efficiency that were ever reported in literature for both laboratory and industrial yeast strains. Interestingly, these engineered xylose and cellobiose utilizing pathways were all host-specific. Therefore, COMPACTER represents a powerful approach to tailor-make metabolic pathways for different strain backgrounds, which is difficult if not impossible to achieve by existing pathway engineering methods.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    'Shotgun DNA synthesis' for the high-throughput construction of large DNA molecules (2012)
    Oxford University Press
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    Publication Date: 2012-10-10
    Description: We developed a highly scalable ‘shotgun’ DNA synthesis technology by utilizing microchip oligonucleotides, shotgun assembly and next-generation sequencing technology. A pool of microchip oligonucleotides targeting a penicillin biosynthetic gene cluster were assembled into numerous random fragments, and tagged with 20 bp degenerate barcode primer pairs. An optimal set of error-free fragments were identified by high-throughput DNA sequencing, selectively amplified using the barcode sequences, and successfully assembled into the target gene cluster.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    Rapid, modular and reliable construction of complex mammalian gene circuits (2013)
    Oxford University Press
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    Publication Date: 2013-09-06
    Description: We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up to 64 kb in size comprising 11 transcription units and 33 basic parts. We show robust gene expression control of multiple transcription units by small molecule inducers in human cells with transient transfection and stable chromosomal integration of these circuits. This framework enables development of complex gene circuits for engineering mammalian cells with unprecedented speed, reliability and scalability and should have broad applicability in a variety of areas including mammalian cell fermentation, cell fate reprogramming and cell-based assays.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    Multiplexing clonality: combining RGB marking and genetic barcoding (2014)
    Oxford University Press
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    Publication Date: 2014-04-15
    Description: RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies.
    Keywords: Synthetic Biology and Assembly Cloning
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    S/MAR sequence confers long-term mitotic stability on non-integrating lentiviral vector episomes without selection (2014)
    Oxford University Press
    Details
    Publication Date: 2014-04-15
    Description: Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting ‘anchoring’ non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for 〉100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo .
    Keywords: Synthetic Biology and Assembly Cloning
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    PaperClip: rapid multi-part DNA assembly from existing libraries (2014)
    Oxford University Press
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    Publication Date: 2014-11-12
    Description: Assembly of DNA ‘parts’ to create larger constructs is an essential enabling technique for bioengineering and synthetic biology. Here we describe a simple method, PaperClip, which allows flexible assembly of multiple DNA parts from currently existing libraries cloned in any vector. No restriction enzymes, mutagenesis of internal restriction sites, or reamplification to add end homology are required. Order of assembly is directed by double stranded oligonucleotides—‘Clips’. Clips are formed by ligation of pairs of oligonucleotides corresponding to the ends of each part. PaperClip assembly can be performed by polymerase chain reaction or by cell extract-mediated recombination. Once multi-use Clips have been prepared, assembly of at least six DNA parts in any order can be accomplished with high efficiency within several hours.
    Keywords: Synthetic Biology and Assembly Cloning
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    Topics: Biology
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    MACE: model based analysis of ChIP-exo (2014)
    Oxford University Press
    Details
    Publication Date: 2014-11-12
    Description: Understanding the role of a given transcription factor (TF) in regulating gene expression requires precise mapping of its binding sites in the genome. Chromatin immunoprecipitation-exo, an emerging technique using exonuclease to digest TF unbound DNA after ChIP, is designed to reveal transcription factor binding site (TFBS) boundaries with near-single nucleotide resolution. Although ChIP-exo promises deeper insights into transcription regulation, no dedicated bioinformatics tool exists to leverage its advantages. Most ChIP-seq and ChIP-chip analytic methods are not tailored for ChIP-exo, and thus cannot take full advantage of high-resolution ChIP-exo data. Here we describe a novel analysis framework, termed MACE (model-based analysis of ChIP-exo) dedicated to ChIP-exo data analysis. The MACE workflow consists of four steps: (i) sequencing data normalization and bias correction; (ii) signal consolidation and noise reduction; (iii) single-nucleotide resolution border peak detection using the Chebyshev Inequality and (iv) border matching using the Gale-Shapley stable matching algorithm. When applied to published human CTCF, yeast Reb1 and our own mouse ONECUT1/HNF6 ChIP-exo data, MACE is able to define TFBSs with high sensitivity, specificity and spatial resolution, as evidenced by multiple criteria including motif enrichment, sequence conservation, direct sequence pileup, nucleosome positioning and open chromatin states. In addition, we show that the fundamental advance of MACE is the identification of two boundaries of a TFBS with high resolution, whereas other methods only report a single location of the same event. The two boundaries help elucidate the in vivo binding structure of a given TF, e.g. whether the TF may bind as dimers or in a complex with other co-factors.
    Keywords: Chromatin and Epigenetics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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    The Effects of Energy Prices on Agricultural Groundwater Extraction from the High Plains Aquifer (2014)
    Oxford University Press
    Details
    Publication Date: 2014-09-02
    Description: We examine the effects of energy prices on groundwater extraction using an econometric model of a farmer's irrigation water pumping decision that accounts for both the intensive and extensive margins. Our results show that energy prices have an effect on both types of margins. Increasing energy prices would affect crop selection decisions, crop acreage allocation decisions, and farmers’ demand for water. Our estimated total marginal effect, which sums the effects on the intensive and extensive margins, suggests that a $1 per million btu increase in the energy price would decrease water extraction by an individual farmer by 5.89 acre-feet per year, a decrease of 3.6 percent of the average annual extraction rate. Our estimated elasticity of water extraction with respect to energy price is –0.26.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q40 - General
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
    Published by Oxford University Press
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    Do Wealth Gains from Land Appreciation Cause Farmers to Expand Acreage or Buy Land? (2014)
    Oxford University Press
    Details
    Publication Date: 2014-09-02
    Description: Recent increases in farm real estate values in the United States have increased farm equity. By exploiting periods of high and low appreciation that caused various increases in wealth for farmers owning various shares of their farmland, we examine whether U.S. grain farmers expanded their acres harvested or acres owned in response to an increase in their land wealth. We find that land wealth had little effect on farm size. However, for similarly-sized farms, a larger ownership share (10 percentage points) led to an increase in the growth of land owned (2 percentage points). Because older farmers own more of the land that they farm, greater land appreciation slows the rate at which younger farmers acquire land relative to older farmers.
    Keywords: Q12 - Micro Analysis of Farm Firms, Farm Households, and Farm Input Markets, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Antagonistic control of a dual-input mammalian gene switch by food additives (2014)
    Oxford University Press
    Details
    Publication Date: 2014-08-15
    Description: Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni , which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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    The role of crop land during economic development: evidence from rural Vietnam (2014)
    Oxford University Press
    Details
    Publication Date: 2014-08-02
    Description: Under economic structural changes, some households intensify farming while others reduce it, resulting in significant changes in landholding. This paper studies the link between such changes in landholding and household well-being during a period of rapid economic transformation in Vietnam. Using a rural household panel data set, we find a U-shaped relationship between landholding and well-being: both accumulating crop land and moving out of farming are associated with higher household income and expenditure. Notably, these relationships are greater in communes that are less developed, suggesting that the benefits of structural transformation may diminish at higher levels of development.
    Keywords: I30 - General, I32 - Measurement and Analysis of Poverty, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation
    Print ISSN: 0165-1587
    Electronic ISSN: 1464-3618
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
    Published by Oxford University Press
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    Identification of a small-molecule ligand of the epigenetic reader protein Spindlin1 via a versatile screening platform (2016)
    Oxford University Press
    Details
    Publication Date: 2016-05-20
    Description: Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein–protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader.
    Keywords: Chromatin and Epigenetics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Multiplex pairwise assembly of array-derived DNA oligonucleotides (2016)
    Oxford University Press
    Details
    Publication Date: 2016-03-19
    Description: While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192–252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131–250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput ‘Dial-Out PCR’ protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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    Dynamics of Farmland Ownership and Leasing: Implications for Young and Beginning Farmers (2016)
    Oxford University Press
    Details
    Publication Date: 2016-05-19
    Description: This study considers the transition into farming and growth of new farmers in U.S. agriculture by examining land ownership and leasing trends. Our approach is to characterize the entire distribution by farmer age and farmer experience rather than using young versus old and beginning versus established farmer categories. We also use a linked-farms longitudinal approach to show trends over time in farmland expansion and contraction. We find that farms operated by older beginning farmers tend to be smaller and do not tend to grow over time. Our results show that it is mostly young farmers as opposed to all beginning farmers who rapidly expand their farm operations after entering agriculture. Our findings inform policy makers about the strategies that young and beginning farmers use to start their businesses and expand over time, and suggest more effective approaches for targeting loan programs to both young and beginning farmers.
    Keywords: Q12 - Micro Analysis of Farm Firms, Farm Households, and Farm Input Markets, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q18 - Agricultural Policy ; Food Policy
    Print ISSN: 2040-5790
    Electronic ISSN: 2040-5804
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
    Published by Oxford University Press
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    Corn Area Response to Local Ethanol Markets in the United States: A Grid Cell Level Analysis (2016)
    Oxford University Press
    Details
    Publication Date: 2016-04-24
    Description: We measure corn and total agricultural area response to the biofuels boom in the United States from 2006 to 2010. Specifically, we use newly available micro-scale grid cell data to test whether a location's corn and total agricultural cultivation rose in response to the capacity of ethanol refineries in their vicinity. Based on these data, acreage in corn and overall agriculture not only grew in already-cultivated areas but also expanded into previously uncultivated areas. Acreage in corn and total agriculture also correlated with proximity to ethanol plants, though the relationship dampened over the time period. A formal estimation of the link between acreage and ethanol refineries, however, must account for the endogenous location decisions of ethanol plants and areas of corn supply. We present historical evidence to support the use of the US railroad network as a valid instrument for ethanol plant locations. Our estimates show that a location's neighborhood refining capacity exerts strong and significant effects on acreage planted in corn and total agricultural acreage. The largest impacts of ethanol plants were felt in locations where cultivation area was relatively low. This high-resolution evidence of ethanol impacts on local agricultural outcomes can inform researchers and policy-makers concerned with crop diversity, environmental sustainability, and rural economic development.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q16 - R&D ; Agricultural Technology ; Agricultural Extension Services
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Exchanging Land for Solidarity: Solidarity Transfers among Voluntarily Resettled and Non-resettled Land-Reform Beneficiaries (2016)
    Oxford University Press
    Details
    Publication Date: 2016-04-24
    Description: Mutual aid among villagers in developing countries is often the only means of insuring against economic shocks. We use "lab-in-the-field experiments" in Cambodian villages to study solidarity in established and newly resettled communities. Our experimental participants were part of an agricultural land-distribution project for which they signed up voluntarily. Half of our sample voluntarily resettled one and a half years before this study. Playing a version of the "solidarity game," we identify the effect of voluntary resettlement on willingness to help anonymous fellow villagers. We find that resettled farmers transfer substantially less money to their fellow villagers than farmers who have not resettled. Our experimental results indicate greater vulnerability on the part of resettled households in the initial years after resettlement.
    Keywords: C93 - Field Experiments, D03 - Behavioral Economics ; Underlying Principles, Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Does the Minimum Lot Size Program Affect Farmland Values? Empirical Evidence Using Administrative Data and Regression Discontinuity Design in Taiwan (2016)
    Oxford University Press
    Details
    Publication Date: 2016-04-24
    Description: This study empirically assesses the causal effect of the minimum lot size program on farmland values in Taiwan. A unique dataset of 4,032 parcels of farmland drawn from administrative foreclosure auction profiles between 2000 and 2008 and regression discontinuity design were applied to cope with the endogeneity issue of land use regulations. The results of the parametric and nonparametric estimations indicate that the minimum lot size program significantly increases farmland value by approximately 18% and 15%, respectively. Moreover, the program effect is more pronounced for farmland located in urban/suburban areas. In the absence of a tax effect and externality resulting from non-agricultural activities, the significant program effect on farmland values is likely to result from the effect of the program on farmland's option value for future development.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, R52 - Land Use and Other Regulations
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    A Novel, Spatiotemporally Explicit Ecological-economic Modeling Procedure for the Design of Cost-effective Agri-environment Schemes to Conserve Biodiversity (2016)
    Oxford University Press
    Details
    Publication Date: 2016-03-31
    Description: Agri-environment schemes (AES) compensate farmers for land use measures that are costly to them but beneficial to biodiversity and the environment. We present an ecological-economic modeling procedure for the design of cost-effective AES to conserve grassland biodiversity, which is applicable to large areas, covers many endangered species and grassland types, and includes several hundred different types of mowing regimes, grazing regimes, and combinations of mowing and grazing regimes as land use measures. The modeling procedure also accounts for the spatial variations in the land use measures' costs and in the effects on species and grassland types. The procedure's main novelty is that it considers variations of the costs and impacts on species and grassland types that arise from different timings of the land use measures. Considering the spatial and the temporal dimension of land use measures makes the modeling procedure spatiotemporally explicit. We demonstrate the power of the modeling procedure by evaluating an existing grassland AES in Saxony, Germany, and identify substantial improvements in terms of cost-effectiveness.
    Keywords: Q15 - Land Ownership and Tenure ; Land Reform ; Land Use ; Irrigation, Q57 - Ecological Economics: Ecosystem Services ; Biodiversity Conservation ; Bioeconomics
    Print ISSN: 0002-9092
    Electronic ISSN: 1467-8276
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition , Economics
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    Substantial DNA methylation differences between two major neuronal subtypes in human brain (2016)
    Oxford University Press
    Details
    Publication Date: 2016-04-08
    Description: The brain is built from a large number of cell types which have been historically classified using location, morphology and molecular markers. Recent research suggests an important role of epigenetics in shaping and maintaining cell identity in the brain. To elucidate the role of DNA methylation in neuronal differentiation, we developed a new protocol for separation of nuclei from the two major populations of human prefrontal cortex neurons—GABAergic interneurons and glutamatergic (GLU) projection neurons. Major differences between the neuronal subtypes were revealed in CpG, non-CpG and hydroxymethylation (hCpG). A dramatically greater number of undermethylated CpG sites in GLU versus GABA neurons were identified. These differences did not directly translate into differences in gene expression and did not stem from the differences in hCpG methylation, as more hCpG methylation was detected in GLU versus GABA neurons. Notably, a comparable number of undermethylated non-CpG sites were identified in GLU and GABA neurons, and non-CpG methylation was a better predictor of subtype-specific gene expression compared to CpG methylation. Regions that are differentially methylated in GABA and GLU neurons were significantly enriched for schizophrenia risk loci. Collectively, our findings suggest that functional differences between neuronal subtypes are linked to their epigenetic specification.
    Keywords: Chromatin and Epigenetics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Standardizing chromatin research: a simple and universal method for ChIP-seq (2016)
    Oxford University Press
    Details
    Publication Date: 2016-04-21
    Description: Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a key technique in chromatin research. Although heavily applied, existing ChIP-seq protocols are often highly fine-tuned workflows, optimized for specific experimental requirements. Especially the initial steps of ChIP-seq, particularly chromatin shearing, are deemed to be exceedingly cell-type-specific, thus impeding any protocol standardization efforts. Here we demonstrate that harmonization of ChIP-seq workflows across cell types and conditions is possible when obtaining chromatin from properly isolated nuclei. We established an ultrasound-based nuclei extraction method (NEXSON: Nuclei EXtraction by SONication) that is highly effective across various organisms, cell types and cell numbers. The described method has the potential to replace complex cell-type-specific, but largely ineffective, nuclei isolation protocols. By including NEXSON in ChIP-seq workflows, we completely eliminate the need for extensive optimization and sample-dependent adjustments. Apart from this significant simplification, our approach also provides the basis for a fully standardized ChIP-seq and yields highly reproducible transcription factor and histone modifications maps for a wide range of different cell types. Even small cell numbers (~10 000 cells per ChIP) can be easily processed without application of modified chromatin or library preparation protocols.
    Keywords: Chromatin and Epigenetics
    Print ISSN: 0305-1048
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    Topics: Biology
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    Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution (2012)
    Oxford University Press
    Details
    Publication Date: 2012-12-14
    Description: Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (K d ) 〈10 pM, representing a ~200-fold improvement in binding affinity over the monomeric aptamers and a ~15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Synthetic mammalian trigger-controlled bipartite transcription factors (2013)
    Oxford University Press
    Details
    Publication Date: 2013-07-16
    Description: Synthetic biology has significantly advanced the design of synthetic control devices, gene circuits and networks that can reprogram mammalian cells in a trigger-inducible manner. Prokaryotic helix-turn-helix motifs have become the standard resource to design synthetic mammalian transcription factors that tune chimeric promoters in a small molecule-responsive manner. We have identified a family of Actinomycetes transcriptional repressor proteins showing a tandem TetR-family signature and have used a synthetic biology-inspired approach to reveal the potential control dynamics of these bi-partite regulators. Daisy-chain assembly of well-characterized prokaryotic repressor proteins such as TetR, ScbR, TtgR or VanR and fusion to either the Herpes simplex transactivation domain VP16 or the Krueppel-associated box domain (KRAB) of the human kox-1 gene resulted in synthetic bi- and even tri-partite mammalian transcription factors that could reversibly program their individual chimeric or hybrid promoters for trigger-adjustable transgene expression using tetracycline (TET), -butyrolactones, phloretin and vanillic acid. Detailed characterization of the bi-partite ScbR-TetR-VP16 (ST-TA) transcription factor revealed independent control of TET- and -butyrolactone-responsive promoters at high and double-pole double-throw (DPDT) relay switch qualities at low intracellular concentrations. Similar to electromagnetically operated mechanical DPDT relay switches that control two electric circuits by a fully isolated low-power signal, TET programs ST-TA to progressively switch from TetR-specific promoter-driven expression of transgene one to ScbR-specific promoter-driven transcription of transgene two while ST-TA flips back to exclusive transgene 1 expression in the absence of the trigger antibiotic. We suggest that natural repressors and activators with tandem TetR-family signatures may also provide independent as well as DPDT-mediated control of two sets of transgenes in bacteria, and that their synthetic transcription-factor analogs may enable the design of compact therapeutic gene circuits for gene and cell-based therapies.
    Keywords: Synthetic Biology and Assembly Cloning
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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    Neural stem cells exposed to BrdU lose their global DNA methylation and undergo astrocytic differentiation (2012)
    Oxford University Press
    Details
    Publication Date: 2012-06-28
    Description: Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) is a halogenated nucleotide of low toxicity commonly used to monitor DNA replication. It is considered a valuable tool for in vitro and in vivo studies, including the detection of the small population of neural stem cells (NSC) in the mammalian brain. Here, we show that NSC grown in self-renewing conditions in vitro , when exposed to BrdU, lose the expression of stem cell markers like Nestin, Sox2 and Pax6 and undergo glial differentiation, strongly up-regulating the astrocytic marker GFAP. The onset of GFAP expression in BrdU exposed NSC was paralleled by a reduced expression of key DNA methyltransferases (DNMT) and a rapid loss of global DNA CpG methylation, as we determined by our specially developed analytic assay. Remarkably, a known DNA demethylating compound, 5-aza-2'-deoxycytidine (Decitabine), had similar effect on demethylation and differentiation of NSC. Since our key findings apply also to NSC derived from murine forebrain, our observations strongly suggest more caution in BrdU uses in stem cells research. We also propose that BrdU and its related substances may also open new opportunities for differentiation therapy in oncology.
    Keywords: Chromatin and Epigenetics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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