How PspGI, catalytic domain of EcoRII and Ecl18kI acquire specificities for different DNA targets

Restriction endonucleases Ecl18kI and PspGI/catalytic domain of EcoRII recognize CCNGG and CCWGG sequences (W stands for A or T), respectively. The enzymes are structurally similar, interact identically with the palindromic CC:GG parts of their recognition sequences and flip the nucleotides at their centers. Specificity for the central nucleotides could be influenced by the strength/stability of the base pair to be disrupted and/or by direct interactions of the enzymes with the flipped bases. Here, we address the importance of these contributions. We demonstrate that wt Ecl18kI cleaves oligoduplexes containing canonical, mismatched and abasic sites in the central position of its target sequence CCNGG with equal efficiencies. In contrast, substitutions in the binding pocket for the extrahelical base alter the Ecl18kI preference for the target site: the W61Y mutant prefers only certain mismatched substrates, and the W61A variant cuts exclusively at abasic sites, suggesting that pocket interactions play a major role in base discrimination. PspGI and catalytic domain of EcoRII probe the stability of the central base pair and the identity of the flipped bases in the pockets. This ‘double check’ mechanism explains their extraordinary specificity for an A/T pair in the flipping position

Central base pair flipping and discrimination by PspGI

PspGI is a representative of a group of restriction endonucleases that recognize a pentameric sequence related to CCNGG. Unlike the previously investigated Ecl18kI, which does not have any specificity for the central base pair, PspGI prefers A/T over G/C in its target site. Here, we present a structure of PspGI with target DNA at 1.7 Å resolution. In this structure, the bases at the center of the recognition sequence are extruded from the DNA and flipped into pockets of PspGI. The flipped thymine is in the usual anti conformation, but the flipped adenine takes the normally unfavorable syn conformation. The results of this and the accompanying manuscript attribute the preference for A/T pairs over G/C pairs in the flipping position to the intrinsically lower penalty for flipping A/T pairs and to selection of the PspGI pockets against guanine and cytosine. Our data show that flipping can contribute to the discrimination between normal bases. This adds a new role to base flipping in addition to its well-known function in base modification and DNA damage repair

POMIAR CZASU MARTWEGO METODĄ DWÓCH ŹRÓDEŁ – OPTYMIZACJA PODZIAŁU CZASU POMIARU

The article presents the analysis of the dead time measurement using two sources for a non-paralyzable detector. It determined the optimum division of count rate measurement time between both source measurement and a single source one. Results of the work can be used to optimize dead time measurement for systems which count photons or particles.W artykule zaprezentowano analizę pomiaru czasu martwego detektora nieparaliżowalnego metodą dwóch źródeł. Wyznaczono optymalny podział czasu pomiaru częstości zliczeń dla pomiaru jednym i dwoma źródłami. Wyniki pracy mogą być wykorzystane do optymalizacji systemów zliczających fotony lub cząstki

TEORIA WZMOCNIENIA JEDNOFOLIOWEGO DETEKTORA Z GAZOWYM POWIELANIEM ELEKTRONÓW

Gain prediction theory of single foil Gas Electron Multiplier detector was developed. Gas electron multiplier (GEM) detector with single foil was developed. Soft X-ray spectra with an energy of 5.9 keV emitted by the isotope Fe-55 were measured. On this basis, the dependence of gain and energy resolution from the detector voltage was determined. The simple theory of gain dependence on various detector parameters was developed. Preliminary results of the study confirmed the potential usefulness of the GEM detector as a substitute for the multiwire proportional chamber.Opracowano teorię wzmocnienia jednofoliowego detektora z gazowym powielaniem elektronów. Opracowano detektor z gazowym powielaniem elektronów z pojedynczą folią. Zmierzono widmo miękkiego promieniowania X, o energii 5,9 keV, emitowanego przez izotop Fe-55. Na tej podstawie wyznaczono zależność wzmocnienia i energetycznej zdolności rozdzielczej od napięcia zasilającego detektor. Opracowano prosta teorią zależności wzmocnienia od różnych parametrów detektora. Wstępne rezultaty badań potwierdzają potencjalną przydatność detektora GEM jako substytutu wielodrutowej komory proporcjonalnej

Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence

Many DNA modification and repair enzymes require access to DNA bases and therefore flip nucleotides. Restriction endonucleases (REases) hydrolyze the phosphodiester backbone within or in the vicinity of the target recognition site and do not require base extrusion for the sequence readout and catalysis. Therefore, the observation of extrahelical nucleotides in a co-crystal of REase Ecl18kI with the cognate sequence, CCNGG, was unexpected. It turned out that Ecl18kI reads directly only the CCGG sequence and skips the unspecified N nucleotides, flipping them out from the helix. Sequence and structure conservation predict nucleotide flipping also for the complexes of PspGI and EcoRII with their target DNAs (/CCWGG), but data in solution are limited and indirect. Here, we demonstrate that Ecl18kI, the C-terminal domain of EcoRII (EcoRII-C) and PspGI enhance the fluorescence of 2-aminopurines (2-AP) placed at the centers of their recognition sequences. The fluorescence increase is largest for PspGI, intermediate for EcoRII-C and smallest for Ecl18kI, probably reflecting the differences in the hydrophobicity of the binding pockets within the protein. Omitting divalent metal cations and mutation of the binding pocket tryptophan to alanine strongly increase the 2-AP signal in the Ecl18kI–DNA complex. Together, our data provide the first direct evidence that Ecl18kI, EcoRII-C and PspGI flip nucleotides in solution

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Semantically Enabled Service-Oriented Architectures: A Manifesto and a Paradigm Shift in Computer Science

After four decades of rapid advances in computing, we are embarking on the greatest leap forward in computing that includes revolutionary changes at all levels of computing from the hardware through the middleware and infrastructure to applications and more importantly in intelligence. This paper outlines a comprehensive framework that integrates two complimentary and revolutionary technical advances, ServiceOriented Architectures (SOA) and Semantic Web, into a single computing architecture, that we call Semantically Enabled Service Oriented Architecture (SESA). While SOA is widely acknowledged for its potential to revolutionize the world of computing, that success depends on resolving two fundamental challenges that SOA does not address, integration, and search or mediation. In a servicesoriented world, billions of services must be discovered and selected based on requirements, then orchestrated and adapted or integrated. SOA depends on but does not address either search or integration. The contribution of this paper is to provide the semanticsbased solution to search and integration that will enable the SOA revolution. The paper provides a vision of the future enabled by our framework that places computing and programming at the services layer and places the real goal of computing, problem solving, in the hands of end users