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  • 1
    Language: English
    In: Journal of molecular biology, 09 December 2011, Vol.414(4), pp.624-37
    Description: Positive feedback in nucleosome modification has been proposed to allow large chromatin regions to exist stably and heritably in distinct expression states. However, modeling has shown that such epigenetic bistability requires that modifying enzymes recruited by nucleosomes are active on distant nucleosomes, potentially allowing uncontrollable spreading of modification. By modeling the silencing of mating-type loci in Saccharomyces cerevisiae, we show that a modification reaction that combines a long-range component and a locally acting component can provide bistability and can be blocked by simple barriers that interrupt the nucleosome chain. We find that robust containment of the silenced region could be achieved by the presence of a number of weak simple barriers in the surrounding chromatin and a limited capacity of the positive feedback reaction. In addition, we show that the state of the silenced region can be regulated by silencer elements acting only on neighboring nucleosomes. Thus, a relatively simple set of nucleosome-modifying enzymes and recognition domains is all that is needed to make chromatin-based epigenetics useful and safe.
    Keywords: Gene Silencing ; Silencer Elements, Transcriptional ; Nucleosomes -- Genetics
    ISSN: 00222836
    E-ISSN: 1089-8638
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States, Feb 19, 2013, Vol.110(8), p.2922(6)
    Description: How distant enhancer elements regulate the assembly of a transcription complex at a promoter remains poorly understood. Here, we use long-range gene regulation by the bacteriophage [lambda] CI protein as a powerful system to examine this process in vivo. A 2.3-kb DNA loop, formed by CI bridging its binding sites at OR and OL, is known already to enhance repression at the lysogenic promoter PRM, located at OR. Here, we show that CI looping also activates PRM by allowing the C-terminal domain of the [alpha] subunit of the RNA polymerase bound at PRM to contact a DNA site adjacent to the distal CI sites at OL. Our results establish OL as a multifaceted enhancer element, able to activate transcription from long distances independently of orientation and position. We develop a physicochemical model of our in vivo data and use it to show that the observed activation is consistent with a simple recruitment mechanism, where the [alpha]-C-terminal domain to DNA contact need only provide ~2.7 kcal/mol of additional binding energy for RNA polymerase. Structural modeling of this complete enhancer-promoter complex reveals how the contact is achieved and regulated, and suggests that distal enhancer elements, once appropriately positioned at the promoter, can function in essentially the same way as proximal promoter elements. www.pnas.org/cgi/doi/ 10.1073/pnas.1221322110
    Keywords: Dna -- Genetic Aspects ; Dna -- Research ; Dna -- Analysis ; Transcription (Genetics) -- Genetic Aspects ; Transcription (Genetics) -- Research ; Transcription (Genetics) -- Analysis ; Promoters (Genetics) -- Genetic Aspects ; Promoters (Genetics) -- Research ; Promoters (Genetics) -- Analysis
    ISSN: 0027-8424
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  • 3
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 07 January 2014, Vol.111(1), pp.349-54
    Description: Efficient and specific interactions between proteins bound to the same DNA molecule can be dependent on the length of the DNA tether that connects them. Measurement of the strength of this DNA tethering effect has been largely confined to short separations between sites, and it is not clear how it contributes to long-range DNA looping interactions, such as occur over separations of tens to hundreds of kilobase pairs in vivo. Here, gene regulation experiments using the LacI and λ CI repressors, combined with mathematical modeling, were used to quantitate DNA tethering inside Escherichia coli cells over the 250- to 10,000-bp range. Although LacI and CI loop DNA in distinct ways, measurements of the tethering effect were very similar for both proteins. Tethering strength decreased with increasing separation, but even at 5- to 10-kb distances, was able to increase contact probability 10- to 20-fold and drive efficient looping. Tethering in vitro with the Lac repressor was measured for the same 600-to 3,200-bp DNAs using tethered particle motion, a single molecule technique, and was 5- to 45-fold weaker than in vivo over this range. Thus, the enhancement of looping seen previously in vivo at separations below 500 bp extends to large separations, underlining the need to understand how in vivo factors aid DNA looping. Our analysis also suggests how efficient and specific looping could be achieved over very long DNA separations, such as what occurs between enhancers and promoters in eukaryotic cells.
    Keywords: Tpm ; J Factor ; Promoter-Enhancer ; DNA, Bacterial -- Genetics ; Escherichia Coli -- Genetics ; Lac Repressors -- Genetics ; Repressor Proteins -- Genetics ; Viral Regulatory and Accessory Proteins -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 4
    Language: English
    In: Educational and Psychological Measurement, Oct, 2013, Vol.73(5), p.854-874
    Description: The progressive-restricted standard error (PR-SE) exposure control procedure was compared to three procedures used in computerized adaptive testing, the randomesque, SympsonuHetter (SH), and no exposure control methods. It was found that the results obtained by PR-SE are similar and acceptable compared to the other exposure control procedures while vastly improving on item pool usage.
    Keywords: Item Response Theory -- Analysis ; Computer Education -- Evaluation
    ISSN: 0013-1644
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  • 5
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 19 February 2013, Vol.110(8), pp.2922-7
    Description: How distant enhancer elements regulate the assembly of a transcription complex at a promoter remains poorly understood. Here, we use long-range gene regulation by the bacteriophage λ CI protein as a powerful system to examine this process in vivo. A 2.3-kb DNA loop, formed by CI bridging its binding sites at OR and OL, is known already to enhance repression at the lysogenic promoter PRM, located at OR. Here, we show that CI looping also activates PRM by allowing the C-terminal domain of the α subunit of the RNA polymerase bound at PRM to contact a DNA site adjacent to the distal CI sites at OL. Our results establish OL as a multifaceted enhancer element, able to activate transcription from long distances independently of orientation and position. We develop a physicochemical model of our in vivo data and use it to show that the observed activation is consistent with a simple recruitment mechanism, where the α-C-terminal domain to DNA contact need only provide ∼2.7 kcal/mol of additional binding energy for RNA polymerase. Structural modeling of this complete enhancer-promoter complex reveals how the contact is achieved and regulated, and suggests that distal enhancer elements, once appropriately positioned at the promoter, can function in essentially the same way as proximal promoter elements.
    Keywords: Enhancer Elements, Genetic ; Transcriptional Activation ; Bacteriophage Lambda -- Genetics ; DNA, Viral -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 6
    Language: English
    In: Plant and Soil, 2014, Vol.382(1), pp.17-30
    Description: Background and aims: Liming is considered normal agricultural practise for remediating soil acidity and improving crop productivity; however recommended lime applications can reduce yield. We tested the hypothesis that elevated xylem sap Ca super(2+) limited gas exchange of Phaseolus vulgaris L. and Pisum sativum L. plants that exhibited reduced shoot biomass and leaf area when limed. Methods: We used Scholander and whole-plant pressure chamber techniques to collect root and leaf xylem sap, a calcium-specific ion-selective electrode to measure xylem sap Ca super(2+), infra-red gas analysis to measure gas exchange of limed and unlimed (control) plants, and a detached leaf transpiration bioassay to determine stomatal sensitivity to Ca super(2+). Results: Liming reduced shoot biomass, leaf area and leaf gas exchange in both species. Root xylem sap Ca super(2+) concentration was only increased in P. vulgaris and not in P. sativum. Detached leaves of both species required 5 mM Ca super(2+) supplied to via the transpiration stream to induce stomatal closure, however, maximum in vivo xylem sap Ca super(2+) concentrations of limed plants was only 1.7 mM and thus not high enough to influence stomata. Conclusion: We conclude that an alternative xylem-borne antitranspirant other than Ca super(2+) decreases gas exchange of limed plants.
    Keywords: Calcium ; Lime ; Pisum sativum ; Phaseolus vulgaris ; Stomatal conductance ; Xylem sap
    ISSN: 0032-079X
    E-ISSN: 1573-5036
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  • 7
    Language: English
    In: Reliability Engineering and System Safety, 2012, Vol.98(1), pp.7-23
    Description: Many safety-critical aircraft functions are software-enabled. Airborne software must be audited and approved by the aerospace certification authorities prior to deployment. The auditing process is time-consuming, and its outcome is unpredictable, due to the criticality and complex nature of airborne software. To ensure that the engineering of airborne software is systematically regulated and is auditable, certification authorities mandate compliance with safety standards that detail industrial best practice. This paper reviews existing practices in software safety certification. It also explores how software safety audits are performed in the civil aerospace domain. The paper then proposes a statistical method for supporting software safety audits by collecting and analysing data about the software throughout its lifecycle. This method is then empirically evaluated through an industrial case study based on data collected from 9 aerospace projects covering 58 software releases. The results of this case study show that our proposed method can help the certification authorities and the software and safety engineers to gain confidence in the certification readiness of airborne software and predict the likely outcome of the audits. The results also highlight some confidentiality issues concerning the management and retention of sensitive data generated from safety-critical projects.
    Keywords: Software Safety ; Certification ; Airborne Software ; Do178b ; Safety Standards ; Safety Requirements ; Engineering
    ISSN: 0951-8320
    E-ISSN: 1879-0836
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  • 8
    In: Journal of Experimental Botany, 2012, Vol. 63(9), pp.3415-3428
    Description: The use of soil and irrigation water with a high content of soluble salts is a major limiting factor for crop productivity in the semi-arid areas of the world. While important physiological insights about the mechanisms of salt tolerance in plants have been gained, the transfer of such knowledge into crop improvement has been limited. The identification and exploitation of soil microorganisms (especially rhizosphere bacteria and mycorrhizal fungi) that interact with plants by alleviating stress opens new alternatives for a pyramiding strategy against salinity, as well as new approaches to discover new mechanisms involved in stress tolerance. Although these mechanisms are not always well understood, beneficial physiological effects include improved nutrient and water uptake, growth promotion, and alteration of plant hormonal status and metabolism. This review aims to evaluate the beneficial effects of soil biota on the plant response to saline stress, with special reference to phytohormonal signalling mechanisms that interact with key physiological processes to improve plant tolerance to the osmotic and toxic components of salinity. Improved plant nutrition is a quite general beneficial effect and may contribute to the maintenance of homeostasis of toxic ions under saline stress. Furthermore, alteration of crop hormonal status to decrease evolution of the growth-retarding and senescence-inducing hormone ethylene (or its precursor 1-aminocyclopropane-1-carboxylic acid), or to maintain source–sink relations, photosynthesis, and biomass production and allocation (by altering indole-3-acetic acid and cytokinin biosynthesis) seem to be promising target processes for soil biota-improved crop salt tolerance.
    Keywords: Ion Homeostasis ; Mycorrhizae ; Phytohormones ; Rhizobacteria ; Salinity ; Source–Sink Relations
    ISSN: 0022-0957
    E-ISSN: 1460-2431
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  • 9
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 21 October 2014, Vol.111(42), pp.E4449-57
    Description: Eukaryotic gene regulation involves complex patterns of long-range DNA-looping interactions between enhancers and promoters, but how these specific interactions are achieved is poorly understood. Models that posit other DNA loops--that aid or inhibit enhancer-promoter contact--are difficult to test or quantitate rigorously in eukaryotic cells. Here, we use the well-characterized DNA-looping proteins Lac repressor and phage λ CI to measure interactions between pairs of long DNA loops in E. coli cells in the three possible topological arrangements. We find that side-by-side loops do not affect each other. Nested loops assist each other's formation consistent with their distance-shortening effect. In contrast, alternating loops, where one looping element is placed within the other DNA loop, inhibit each other's formation, thus providing clear support for the loop domain model for insulation. Modeling shows that combining loop assistance and loop interference can provide strong specificity in long-range interactions.
    Keywords: Lac Repressor ; Lambda Ci ; Statistical Mechanical Modeling ; Tethered Particle Motion ; DNA, Bacterial -- Chemistry ; Escherichia Coli -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 10
    Language: English
    In: The Journal of biological chemistry, 14 November 2014, Vol.289(46), pp.32094-108
    Description: The lysogeny promoting protein CII from bacteriophage 186 is a potent transcriptional activator, capable of mediating at least a 400-fold increase in transcription over basal activity. Despite being functionally similar to its counterpart in phage λ, it shows no homology at the level of protein sequence and does not belong to any known family of transcriptional activators. It also has the unusual property of binding DNA half-sites that are separated by 20 base pairs, center to center. Here we investigate the structural and functional properties of CII using a combination of genetics, in vitro assays, and mutational analysis. We find that 186 CII possesses two functional domains, with an independent activation epitope in each. 186 CII owes its potent activity to activation mechanisms that are dependent on both the σ(70) and α C-terminal domain (αCTD) components of RNA polymerase, contacting different functional domains. We also present evidence that like λ CII, 186 CII is proteolytically degraded in vivo, but unlike λ CII, 186 CII proteolysis results in a specific, transcriptionally inactive, degradation product with altered self-association properties.
    Keywords: Bacterial Transcription ; Bacteriophage ; Gene Transcription ; Mutational Screen ; Promoter ; Protein Structure-Function ; Proteolysis ; RNA Polymerase ; Promoter Regions, Genetic ; Transcription Factors -- Genetics ; Viral Proteins -- Genetics
    E-ISSN: 1083-351X
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