Simian virus 40 large T-antigen G-quadruplex DNA helicase inhibition by G-quadruplex DNA-interactive agents

On the basis of growing evidence for G-quadruplex DNA structures in genomic DNA and the presumed need to resolve these structures for DNA replication, the G-quadruplex DNA unwinding ability of a prototypical replicative helicase, SV40 large T-antigen (T-ag), was investigated. Here, we demonstrate that this G-quadruplex helicase activity is robust and comparable to the duplex helicase activity of T-ag. Analysis of the SV40 genome demonstrates the presence of sequences that may form intramolecular G-quadruplexes, which are the presumed natural substrates for the G-quadruplex helicase activity of T-ag. A number of G-quadruplex-interactive agents as well as new perylene diimide (PDI) derivatives have been investigated as inhibitors of both the G-quadruplex and the duplex DNA helicase activities of T-ag. A unique subset of these G-quadruplex-interactive agents inhibits the G-quadruplex DNA unwinding activity of T-ag, relative to those reported to inhibit G-quadruplex DNA unwinding by RecQ-family helicases. We also find that certain PDIs are both potent and selective inhibitors of the G-quadruplex DNA helicase activity of T-ag. Surface plasmon resonance and fluorescence spectroscopic G-quadruplex DNA binding studies of these T-ag G-quadruplex helicase inhibitors have been carried out, demonstrating the importance of attributes in addition to binding affinity for G-quadruplex DNA that may be important for inhibition. The identification of potent and selective inhibitors of the G-quadruplex helicase activity of T-ag provides tools for probing the specific role of this activity in SV40 replication.     

Model for T-antigen-dependent melting of the simian virus 40 core origin based on studies of the interaction of the beta-hairpin with DNA

The interaction of simian virus 40 (SV40) T antigen (T-ag) with the viral origin has served as a model for studies of site-specific recognition of a eukaryotic replication origin and the mechanism of DNA unwinding. These studies have revealed that a motif termed the "beta-hairpin" is necessary for assembly of T-ag on the SV40 origin. Herein it is demonstrated that residues at the tip of the "beta-hairpin" are needed to melt the origin-flanking regions and that the T-ag helicase domain selectively assembles around one of the newly generated single strands in a manner that accounts for its 3'-to-5' helicase activity. Furthermore, T-ags mutated at the tip of the "beta-hairpin" are defective for oligomerization on duplex DNA; however, they can assemble on hybrid duplex DNA or single-stranded DNA (ssDNA) substrates provided the strand containing the 3' extension is present. Collectively, these experiments indicate that residues at the tip of the beta-hairpin generate ssDNA in the core origin and that the ssDNA is essential for subsequent oligomerization events.     

Leitlinien zur molekulargenetischen Diagnostik: Fragiles-X und Fragiles-X assoziiertes Tremor/Ataxie Syndrom

Ohne Zusammenfassung

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Individual variability of behavioural responses by Wandering Albatrosses (Diomedea exulans) to human disturbance

Sub-Antarctic Marion Island has had a permanent research station for 50 years and the islands Wandering Albatrosses have been intensively studied for 20 years. The reactions of breeding birds to approaches by a human on foot were recorded. Three response variables were calculated: intensity of vocal reaction (IVR), intensity of non-vocal reaction (INR) and overall response index (ORI). At 5 m from the nest, twice as many birds stood and/or vocalised as at 15 m. Nearest neighbour distance, age and gender did not explain individual variability of responses. Study colony birds had higher IVR scores than non-study colony birds; birds at colonies closest to the station had the highest ORI scores. A better breeding record was associated with lower IVR and ORI scores, but a causative relationship remains to be demonstrated. A minimum viewing distance of 25 m is recommended for breeding Wandering Albatrosses.

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Semi-automated, Membrane-Based Protocol for DNA Isolation from Plants

Many plant species are considered difficult for DNA isolation due to their high concentrations of secondary metabolites such as polysaccharides and polyphenols. Several protocols have been developed to overcome this problem, but they are typically time-consuming, not scalable for high throughput and not compatible with automation. Although a variety of commercial kits are available for plant DNA isolation, their cost is high and these kits usually have limited taxonomic applicability. In a previous study we developed an inexpensive automation-friendly protocol for DNA extraction from animal tissues. Here we demonstrate that a similar protocol allows DNA isolation from plants.

neo-Clerodane diterpenoids from Ajuga: structural elucidation and biological activity

The presence of several types of allelochemicals has been reported from Ajuga, a Labiatae genus comprising more than 40 species of wide distribution in extratropical regions of both hemispheres. The genus is of great medicinal and economic importance and among the biological properties of the secondary metabolites, the antifeedant activity against pest insects appears to be related to the presence of neo-clerodane type diterpenes. This review focuses on the isolation and structural elucidation of this type of compounds from Ajuga species and the hemisynthetic compounds of closely related structure obtained. The reported biological activity of crude extracts and isolated diterpenes will be briefly commented.

Hennigian Phylogenetic Systematics and the ‘Groundplan’ vs. ‘Post-Groundplan’ Approaches: A Reply to Kukalová-Peck

The recent contribution by Jarmila Kukalová-Peck on Hennigian phylogenetics and hexapod limb evolution is critically evaluated.

Patterns of genome size diversity in the ray-finned fishes

The ray-finned fishes make up about half of all vertebrate diversity and are by far the best represented group in the Animal Genome Size Database. However, they have traditionally been the least well investigated among vertebrates in terms of patterns and consequences of genome size diversity. This article synthesizes and expands upon existing information about genome size diversity in ray-finned fishes. Specifically, compiled data from the Animal Genome Size Database and FishBase are used to examine the potential patterns of interspecific genome size variability according to ecology, environment, morphology, growth, physiology, reproduction, longevity, and taxonomic diversity. Polyploidy and haploid genome sizes are considered separately, revealing differences in their respective consequences. This represents the most comprehensive summary of fish genome size diversity presented to date, and highlights areas of particular interest to investigate as more data become available.

Genomic Error-Correcting Codes in the Living World

This paper is intended to complement our previous works on the necessary existence of error-correcting codes endowing genomes with the ability of being regenerated, not merely copied. It sketchily recalls some fundamental definitions and results of information theory and error-correcting codes; provides an overview of our research; shows that the disjunction of replication and regeneration enlightens the divide between germinal and somatic cells; suggests that some phenomena referred to as epigenetic may possibly find an explanation within the framework of error-correcting codes; points out some difficulties, especially those related to sexual reproduction; criticizes the template-replication paradigm, and prompts geneticists to become familiar with information theory.

Is it a revolution?

Jablonka and Lamb's claim that evolutionary biology is undergoing a ‘revolution’ is queried. But the very concept of revolutionary change has uncertain application to a field organized in the manner of contemporary biology. The explanatory primacy of sequence properties is also discussed.

Gridcast—a next generation broadcast infrastructure?

Gridcast is an R&D project investigating grid ideas and technologies in the broadcasting technical infrastructure. In this paper I discuss the business and technical issues in building infrastructures to support broadcasters and outline the structure of the Gridcast grid-based service oriented architecture for broadcasting playout support.

A single operon-encoded form of the acetyl-CoA decarbonylase/synthase multienzyme complex responsible for synthesis and cleavage of acetyl-CoA in <Emphasis Type="Italic">Methanosarcina thermophila</Emphasis>

Methanogens growing on C-1 substrates synthesize 2-carbon acetyl groups in the form of acetyl-CoA for carbon assimilation using the multienzyme complex acetyl-CoA decarbonylase/synthase (ACDS) which contains five different subunits encoded within an operon. In species growing on acetate ACDS also functions to cleave the acetate C-C bond for energy production by methanogenesis. A number of species of Methanosarcina that are capable of growth on either C-1 compounds or acetate contain two separate ACDS operons, and questions have been raised about whether or not these operons play separate roles in acetate synthesis and cleavage. Methanosarcina thermophila genomic DNA was analyzed for the presence of two ACDS operons by PCR amplifications with different primer pairs, restriction enzyme analyses, DNA sequencing and Southern blot analyses. A single ACDS operon was identified and characterized, with no evidence for more than one. MALDI mass spectrometric analyses were carried out on ACDS preparations from methanol- and acetate-grown cells. Peptide fragmentation patterns showed that the same ACDS subunits were present regardless of growth conditions. The evidence indicates that a single form of ACDS is used both for acetate cleavage during growth on acetate and for acetate synthesis during growth on C-1 substrates. Electronic Supplementary Material  Supplementary material is available for this article at

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Information,complexity and generative replication

The established definition of replication in terms of the conditions of causality, similarity and information transfer is very broad. We draw inspiration from the literature on self-reproducing automata to strengthen the notion of information transfer in replication processes. To the triple conditions of causality, similarity and information transfer, we add a fourth condition that defines a “generative replicator” as a conditional generative mechanism, which can turn input signals from an environment into developmental instructions. Generative replication must have the potential to enhance complexity, which in turn requires that developmental instructions are part of the information that is transmitted in replication. Demonstrating the usefulness of the generative replicator concept in the social domain, we identify social generative replicators that satisfy all of the four proposed conditions.

Hierarchical analysis of piecewise affine models of gene regulatory networks

A key point in the analysis of dynamical models of biological systems is to handle systems of relatively high dimensions. In the present paper we propose a method to hierarchically organize a certain type of piecewise affine (PWA) differential systems. This specific class of systems has been extensively studied for the past few years, as it provides a good framework to model gene regulatory networks. The method, shown on several examples, allows a qualitative analysis of the asymptotic behavior of a PWA system, decomposing it into several smaller subsystems. This technique, based on the well-known strongly connected components decomposition, is not new. However, its adaptation to the non-smooth PWA differential equations turns out to be quite relevant because of the strong discrete structure underlying these equations. Its biological relevance is shown on a 7-dimensional PWA system modeling the gene network responsible for the carbon starvation response in Escherichia coli.

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Helicase Loading at Chromosomal Origins of Replication

Loading of the replicative DNA helicase at origins of replication is of central importance in DNA replication. As the first of the replication fork proteins assemble at chromosomal origins of replication, the loaded helicase is required for the recruitment of the rest of the replication machinery. In this work, we review the current knowledge of helicase loading at Escherichia coli and eukaryotic origins of replication. In each case, this process requires both an origin recognition protein as well as one or more additional proteins. Comparison of these events shows intriguing similarities that suggest a similar underlying mechanism, as well as critical differences that likely reflect the distinct processes that regulate helicase loading in bacterial and eukaryotic cells.Replicative DNA helicases are ring-shaped molecules with a central cavity through which DNA passes as they unwind DNA. Their loading at replication origins is a critical and highly regulated event in chromosomal replication. The DNA helicase is the first of the replication fork proteins recruited to and loaded onto origins of replication, and the loaded helicase is required for the recruitment of the rest of the replication machinery (Remus and Diffley 2009; Kaguni 2011). Indeed, the replicative DNA helicase links the replication machinery to the parental DNA (O’Donnell 2006). In Escherichia coli cells, the DnaB replicative helicase binds to primase (DnaG) and the sliding clamp loader, which in turn binds the DNA polymerases. Although the polymerases are also linked to the template DNA by sliding clamps, when these interactions are broken, the polymerases’ association with the sliding clamp loader and the helicase keeps them at the site of replication. The interactions that tether the DNA polymerases to the eukaryotic replication fork are less clear but very likely involve direct and indirect interactions with the Mcm2–7 replicative helicase (Calzada et al. 2005).Helicase loading is carefully regulated to control the location and frequency of replication initiation. In eukaryotic cells, helicase loading is tightly restricted to the G₁ phase of the cell cycle. This constraint is a key part of the mechanisms that ensure that no origin can initiate more than once per cell cycle (Siddiqui et al. 2013; Zielke et al. 2013). In addition, the sites of eukaryotic replicative helicase loading define the potential sites of replication initiation in the cell (but not all loaded helicases are used during a given S phase [Rhind and Gilbert 2012]). Although the central regulated event in bacterial chromosome duplication is the recruitment of the ATP-bound initiator protein DnaA (Skarstad and Katayama 2013), the loading of the replicative helicase represents a key committed step during initiation.Here we will discuss the mechanism of helicase loading in bacteria and eukaryotic cells. Much of the discussion will focus on studies in the bacterium E. coli, the yeast Saccharomyces cerevisiae, and the frog Xenopus laevis, in which the events of helicase loading are best understood. Comparison of these mechanisms shows important similarities and differences between the domains of life. In both bacteria and eukaryotic cells, multiple AAA⁺ proteins use ATP binding and hydrolysis to direct helicase loading and both helicases are initially loaded in an inactive form. On the other hand, the eukaryotic helicase is loaded around double-stranded DNA (dsDNA) and as a double hexamer, whereas the bacterial helicase is loaded around single-stranded DNA (ssDNA) as a single hexamer. These distinctions are very likely due to the very different regulatory mechanisms of DNA replication in bacteria and eukaryotic cells.Before describing the process of helicase loading, it is relevant to know that the essential function of replicative helicases is to unwind the parental duplex DNA using the energy provided by the hydrolysis of a nucleoside triphosphate (Patel et al. 2011). Replicative DNA polymerases then copy each parental DNA strand to duplicate the genome. That replicative DNA helicases are hexameric (and sometimes heptameric) structures that have a positively charged, central channel provides a framework for how these molecular machines work. Several models that describe the mechanism of unwinding have been considered. One is the steric or strand-exclusion model, in which the helicase excludes one strand of DNA while the other passes through the central cavity as the enzyme moves. Because these enzymes can bind and translocate on duplex DNA without unwinding (Kaplan et al. 2003), two additional models have been proposed. In the ploughshare model, duplex DNA enters the central cavity and exits as unwound DNA by virtue of a domain/protein that acts as a ploughshare or pin, which disrupts the hydrogen bonds of DNA as it is pumped through the enzyme (reviewed in Takahashi et al. 2005). A second model, the DNA-pumping model, was proposed on the basis of the double-hexameric forms of SV40, Mcm2–7, and other replicative helicases (Mastrangelo et al. 1989; Remus et al. 2009). This model proposes that the two helicases pump duplex DNA toward one another, resulting in torsional strain that forces the two strands apart, at which point they exit the central channel as two ssDNA loops. Current evidence supports the steric exclusion model (Jezewska et al. 1998b; Kaplan 2000; Galletto et al. 2004; Fu et al. 2011). Of interest, the E. coli enzyme moves in the 5′-to-3′ direction relative to the engaged ssDNA that passes through the central cavity, whereas archaeal and eukaryotic enzymes move in the 3′-to-5′ direction.

Table 1.

Replicative DNA helicases of free-living organisms are hexameric

Consistency principle in biological dynamical systems

We propose a principle of consistency between different hierarchical levels of biological systems. Given a consistency between molecule replication and cell reproduction, universal statistical laws on cellular chemical abundances are derived and confirmed experimentally. They include a power law distribution of gene expressions, a lognormal distribution of cellular chemical abundances over cells, and embedding of the power law into the network connectivity distribution. Second, given a consistency between genotype and phenotype, a general relationship between phenotype fluctuations by genetic variation and isogenic phenotypic fluctuation by developmental noise is derived. Third, we discuss the chaos mechanism for stem cell differentiation with autonomous regulation, resulting from a consistency between cell reproduction and growth of the cell ensemble.

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