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101.
DNA binding and phasing analyses of Tn5 transposase and a monomeric variant. 总被引:5,自引:3,他引:2 下载免费PDF全文
Both full-length Tn 5 transposase and a COOH-terminal truncated monomeric form of the protein,n369, have been shown to specifically bind end sequences at comparable affinities. In addition, both proteins distort the target sequence in a similar manner, as determined by a circular permutation assay. In this study,nEK54, a derivative ofn369 with a single amino acid substitution that significantly enhances binding activity, is used in further binding and bending studies along with full-length transposase. Phasing analysis has shown that distortion of the end sequences upon binding of full-length transposase and nEK54 protein is due in part to a protein-induced bend oriented towards the major groove. Because the center of transposase-induced bending maps to the extreme leftward end of the 19 bp consensus sequence, we examined the possibility that optimal protein binding requires additional upstream nucleotide contacts. Experiments presented here show that 9-10 nucleotides are needed upstream of +1 of the 19 bp sequence for efficient binding and this requirement can be met by either single-stranded or double-stranded DNA. 相似文献
102.
George M. Giambaşu Tyler Luchko Daniel Herschlag Darrin M. York David A. Case 《Biophysical journal》2014
The ionic atmosphere around nucleic acids remains only partially understood at atomic-level detail. Ion counting (IC) experiments provide a quantitative measure of the ionic atmosphere around nucleic acids and, as such, are a natural route for testing quantitative theoretical approaches. In this article, we replicate IC experiments involving duplex DNA in NaCl(aq) using molecular dynamics (MD) simulation, the three-dimensional reference interaction site model (3D-RISM), and nonlinear Poisson-Boltzmann (NLPB) calculations and test against recent buffer-equilibration atomic emission spectroscopy measurements. Further, we outline the statistical mechanical basis for interpreting IC experiments and clarify the use of specific concentration scales. Near physiological concentrations, MD simulation and 3D-RISM estimates are close to experimental results, but at higher concentrations (>0.7 M), both methods underestimate the number of condensed cations and overestimate the number of excluded anions. The effect of DNA charge on ion and water atmosphere extends 20–25 Å from its surface, yielding layered density profiles. Overall, ion distributions from 3D-RISMs are relatively close to those from corresponding MD simulations, but with less Na+ binding in grooves and tighter binding to phosphates. NLPB calculations, on the other hand, systematically underestimate the number of condensed cations at almost all concentrations and yield nearly structureless ion distributions that are qualitatively distinct from those generated by both MD simulation and 3D-RISM. These results suggest that MD simulation and 3D-RISM may be further developed to provide quantitative insight into the characterization of the ion atmosphere around nucleic acids and their effect on structure and stability. 相似文献
103.
Stepwise cis-Regulatory Changes in ZCN8 Contribute to Maize Flowering-Time Adaptation 总被引:1,自引:0,他引:1
104.
Factors influencing above‐ground and soil seed bank vegetation diversity at different scales in a quasi‐Mediterranean ecosystem 下载免费PDF全文
Matthew P. Chick Craig R. Nitschke Janet S. Cohn Trent D. Penman Alan York 《植被学杂志》2018,29(4):684-694
Questions
Are factors influencing plant diversity in a fire‐prone Mediterranean ecosystem of southeast Australia scale‐dependent?Location
Heathy woodland, Otways region, Victoria, southeast AustraliaMethods
We measured patterns of above‐ground and soil seed bank vegetation diversity and associated them with climatic, biotic, edaphic, topographic, spatial and disturbance factors at multiple scales (macro to micro) using linear mixed effect and generalized dissimilarity modelling.Results
At the macro‐scale, we found species richness above‐ground best described by climatic factors and in the soil seed bank by disturbance factors. At the micro‐scale we found species richness best described above‐ground and in the soil seed bank by disturbance factors, in particular time‐since‐last‐fire. We found variance in macro‐scale β‐diversity (species turnover) best explained above‐ground by climatic and disturbance factors and in the soil seed bank by climatic and biotic factors.Conclusions
Regional climatic gradients interact with edaphic factors and fire disturbance history at small spatial scales to influence species richness and turnover in the studied ecosystem. Current fire management regimes need to incorporate key climatic–disturbance–diversity interactions to maintain floristic diversity in the studied system.105.
At least three perspectives—industrial ecology (IE), ecological modernization theory (EMT), and the “environmental Kuznets curve” (EKC)—emphasize the potential for sustainability via refinements in production systems that dramatically reduce the environmental impacts of economic development. Can improvements in efficiency counterbalance environmental impacts stemming from the scale of production? To address this question we analyze cross‐national variation in the ecological footprint (EF) per unit of gross domestic product (GDP). The EF is a widely recognized indicator of human pressure on the environment. The EF of a nation is the amount of land area that would be required to produce the resources it consumes and to absorb the wastes it generates. The most striking finding of our analyses is that there is limited variation across nations in EF per unit of GDP. This indicates limited plasticity in the levels of EF intensity or eco‐efficiency among nations, particularly among affluent nations. EF intensity is lowest (ecoefficiency is highest) in affluent nations, but the level of efficiency in these nations does not appear to be of sufficient magnitude to compensate for their large productive capacities. These results suggest that modernization and economic development will be insufficient, in themselves, to bring about the ecological sustainability of societies. 相似文献
106.
107.
Simple and efficient generation in vitro of nested deletions and inversions: Tn5 intramolecular transposition. 总被引:2,自引:0,他引:2 下载免费PDF全文
We have exploited the intramolecular transposition preference of the Tn 5 in vitro transposition system to test its effectiveness as a tool for generation of nested families of deletions and inversions. A synthetic transposon was constructed containing an ori, an ampicillin resistance (Ampr) gene, a multi-cloning site (MCS) and two hyperactive end sequences. The donor DNA that adjoins the transposon contains a kanamycin resistance (Kanr) gene. Any Amprreplicating plasmid that has undergone a transposition event (Kans) will be targeted primarily to any insert in the MCS. Two different size targets were tested in the in vitro system. Synthetic transposon plasmids containing either target were incubated in the presence of purified transposase (Tnp) protein and transformed. Transposition frequencies (Ampr/Kans) for both targets were found to be 30-50%, of which >95% occur within the target sequence, in an apparently random manner. By a conservative estimate 10(5) or more deletions/inversions within a given segment of DNA can be expected from a single one-step 20 microl transposition reaction. These nested deletions can be used for structure-function analysis of proteins and for sequence analysis. The inversions provide nested sequencing templates of the opposite strand from the deletions. 相似文献
108.
109.
Effect of alkylguanidines and alkylamines on trypsin catalysis 总被引:4,自引:0,他引:4
110.
Jürgen Hartleb York Damm Rüdiger Arndt Enno Christophers Eggert Stockfleth 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,727(1-2)
5-S-Cysteinyldopa (5-SCD) in plasma and urine was determined by means of a newly developed method. This method incorporates optimized conditions for blood collection and storage, as well as a new extraction and separation technique, required for the strong oxidation and light sensitive 5-SCD. The new aspects of the method are the following: immediate centrifugation and freezing of the samples after blood collection, fully automatical solid-phase extraction (SPE) with phenylboronic acid (PBA) cartridges and immediate HPLC injection of the eluate, nearly complete exclusion of light and air–oxygen during extraction, constant sample cooling, use of the more suitable internal standard 5-S-
-cysteinyldopa and easy, sensitive and selective HPLC conditions (RP18-column with isocratic separation and electrochemical detection). The method has a linear range from 0.25 to 50 μg l−1 and 25 to 5000 μg l−1 for plasma and urine samples, respectively, a limit of detection of 0.17 μg l−1, intra-assay variabilities from 1.7 to 3.6%, inter-assay variabilities from 4.0 to 18.3% and an average relative recovery of 103.5% for plasma and 105.4% for urine samples. In our study the measured 5-SCD concentrations of patients with melanomas at various stages correlated better with their clinical pictures than described in literature up to date. The results were obtained in comparison to patients with other skin tumors and in comparison to healthy control persons. 相似文献