Conformational and thermodynamic hallmarks of DNA operator site specificity in the copper sensitive operon repressor from Streptomyces lividans

Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoRSl) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoRSl binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5′-TAC/GTA-3′ inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoRSl-DNA interaction suggest selectivity towards the A-DNA-like topology of the G-tracts at the operator site. Such properties are enhanced on protein binding thus enabling the symmetrical binding of two CsoRSl tetramers. Finally, differential binding modes may exist in operator sites having more than one 5′-TAC/GTA-3′ inverted repeat with implications in vivo for a mechanism of modular control. © 2013 The Author(s)

Mapping the B-A conformational transition along plasmid DNA

A simple method is presented to monitor conformational isomerizations along genomic DNA. We illustrate properties of the method with the B-A conformational transition induced by ethanol in linearized pUC19 plasmid DNA. At various ethanol concentrations, the DNA was irradiated with ultraviolet light, transferred to a restriction endonuclease buffer and the irradiated DNA was cleaved by 17 restriction endonucleases. The irradiation damaged DNA and the damage blocked the restrictase cleavage. The amount of uncleaved, i.e. damaged, DNA depended on the concentration of ethanol in a characteristic S-shape way typical of the cooperative B-A transition. The transition beginning and midpoint were determined for each restriction endonuclease. These data map the B-A transition along the whole polylinker of pUC19 DNA and six evenly distributed recognition sequences within the rest of the plasmid. The transition midpoints fell within the B-A transition region of the plasmid simultaneously determined by CD spectroscopy. The present method complements the previous methods used to study the B-A transition. It can be employed to analyze multikilobase regions of genomic DNA whose restriction endonuclease cleavage fragments can be separated and quantified on agarose gels

Tax Havens and their Utilization

Bakalářská práce se zabývá tématem daňových rájů a jejich využití. Dále probírá s tím související problematiku mezinárodního daňového plánování. Je rozdělena na tři části. V té první, teoretické, popisuje základní pojmy objasňující danou problematiku. Následuje analytická část, která se zabývá současným stavem zdanění ve vybraných zemích. Poslední částí této práce je praktická, kde si ukážeme, jak by bylo možné využít daňového ráje a zda by to nabízelo nějakou daňovou výhodu.This bachelor thesis describes the topic of tax havens and their usage. Further it describes issues of international tax planning. The thesis is divided into three parts. The first one, theoretical, explains basic concepts of this issue. Analytical part follows with description of present state of taxation in selected countries. The last section of this thesis shows some uses of tax havens and whether or not it offers tax advantages.

Circular dichroism and conformational polymorphism of DNA

Here we review studies that provided important information about conformational properties of DNA using circular dichroic (CD) spectroscopy. The conformational properties include the B-family of structures, A-form, Z-form, guanine quadruplexes, cytosine quadruplexes, triplexes and other less characterized structures. CD spectroscopy is extremely sensitive and relatively inexpensive. This fast and simple method can be used at low- as well as high-DNA concentrations and with short- as well as long-DNA molecules. The samples can easily be titrated with various agents to cause conformational isomerizations of DNA. The course of detected CD spectral changes makes possible to distinguish between gradual changes within a single DNA conformation and cooperative isomerizations between discrete structural states. It enables measuring kinetics of the appearance of particular conformers and determination of their thermodynamic parameters. In careful hands, CD spectroscopy is a valuable tool for mapping conformational properties of particular DNA molecules. Due to its numerous advantages, CD spectroscopy significantly participated in all basic conformational findings on DNA

Reversible DNA i-motif to hairpin switching induced by copper(II) cations

i-Motif DNA structures have previously been utilised for many different nanotechnological applications, but all have used changes in pH to fold the DNA. Herein we describe how copper(ii) cations can alter the conformation of i-motif DNA into an alternative hairpin structure which is reversible by chelation with EDTA

Exchange-Rate Convergence and Entrance to the Eurozone III - Selected Problems of New Member States (in Czech)

The paper contains a general analysis of the exchange rate convergence process of new European Union member states and candidate countries. Following an introduction, we discuss exchange rate strategies applied during periods of economic transition. Emphasis is given to a hypothetical evaluation of the exchange rate criterion. We review the exchange rate strategies of transition countries using an analytical framework similar to the EU member states, and discuss the selected strategies presented in the “Pre-accession Economic Programmes” and convergence programs. In light of the exchange rate experience of EU-15 countries, we summarize the main determinants of exchange rate movement in transition countries. Finally, we discuss factors which will influence ERM2 accession.convergence, exchange rate, monetary policy, transition

Intermediate coherent-incoherent charge transport: DNA as a case study

We study an intermediate quantum coherent-incoherent charge transport mechanism in metal-molecule-metal junctions using B\"uttiker's probe technique. This tool allows us to include incoherent effects in a controlled manner, and thus to study situations in which partial decoherence affects charge transfer dynamics. Motivated by recent experiments on intermediate coherent-incoherent charge conduction in DNA molecules [L. Xiang {\it et al.}, Nature Chem. 7, 221-226 (2015)], we focus on two representative structures: alternating (GC)$_n$ and stacked G$_n$C$_n$ sequences; the latter structure is argued to support charge delocalization within G segments, and thus an intermediate coherent-incoherent conduction. We begin our analysis with a highly simplified 1-dimensional tight-binding model, while introducing environmental effects through B\"uttiker's probes. This minimal model allows us to gain fundamental understanding of transport mechanisms and derive analytic results for molecular resistance in different limits. We then use a more detailed ladder-model Hamiltonian to represent double-stranded DNA structures---with environmental effects captured by B\"uttiker's probes. We find that hopping conduction dominates in alternating sequences, while in stacked sequences charge delocalization (visualized directly through the electronic density matrix) supports significant resonant-ballistic charge dynamics reflected by an even-odd effect and a weak distance dependence for resistance. Our analysis illustrates that lessons learned from minimal models are helpful for interpreting charge dynamics in DNA.Comment: 16 pages, 14 figure

Characteristics of oligonucleotide frequencies across genomes: Conservation versus variation, strand symmetry, and evolutionary implications

One of the objectives of evolutionary genomics is to reveal the genetic information contained in the primordial genome (called the primary genetic information in this paper, with the primordial genome defined here as the most primitive nucleic acid genome for earth’s life) by searching for primitive traits or relics remained in modern genomes. As the shorter a sequence is, the less probable it would be modified during genome evolution. For that reason, some characteristics of very short nucleotide sequences would have considerable chances to persist during billions of years of evolution. Consequently, conservation of certain genomic features of mononucleotides, dinucleotides, and higher-order oligonucleotides across various genomes may exist; some, if not all, of these features would be relics of the primary genetic information. Based on this assumption, we analyzed the pattern of frequencies of mononucleotides, dinucleotides, and higher-order oligonucleotides of the whole-genome sequences from 458 species (including archaea, bacteria, and eukaryotes). Also, we studied the phenomenon of strand symmetry in these genomes. The results show that the conservation of frequencies of some dinucleotides and higher-order oligonucleotides across genomes does exist, and that strand symmetry is a ubiquitous and explicit phenomenon that may contribute to frequency conservation. We propose a new hypothesis for the origin of strand symmetry and frequency conservation as well as for the constitution of early genomes. We conclude that the phenomena of strand symmetry and the pattern of frequency conservation would be original features of the primary genetic information