http://hdl.handle.net/2144/1010 2013-05-19T14:10:24Z 2013-05-19T14:10:24Z Yuzenkova, Yulia Bochkareva, Aleksandra Tadigotla, Vasisht R Roghanian, Mohammad Zorov, Savva Severinov, Konstantin Zenkin, Nikolay http://hdl.handle.net/2144/3355 2012-01-13T07:00:54Z 2010-05-07T00:00:00Z

Stepwise Mechanism for Transcription Fidelity Yuzenkova, Yulia; Bochkareva, Aleksandra; Tadigotla, Vasisht R; Roghanian, Mohammad; Zorov, Savva; Severinov, Konstantin; Zenkin, Nikolay BACKGROUND. Transcription is the first step of gene expression and is characterized by a high fidelity of RNA synthesis. During transcription, the RNA polymerase active centre discriminates against not just non-complementary ribo NTP substrates but also against complementary 2'- and 3'-deoxy NTPs. A flexible domain of the RNA polymerase active centre, the Trigger Loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive. RESULTS. Here we show that transcription fidelity is achieved through a multi-step process. The initial binding in the active centre is the major discrimination step for some non-complementary substrates, although for the rest of misincorporation events discrimination at this step is very poor. During the second step, non-complementary and 2'-deoxy NTPs are discriminated against based on differences in reaction transition state stabilization and partly in general base catalysis, for correct versus non-correct substrates. This step is determined by two residues of the Trigger Loop that participate in catalysis. In the following step, non-complementary and 2'-deoxy NTPs are actively removed from the active centre through a rearrangement of the Trigger Loop. The only step of discrimination against 3'-deoxy substrates, distinct from the ones above, is based on failure to orient the Trigger Loop catalytic residues in the absence of 3'OH. CONCLUSIONS. We demonstrate that fidelity of transcription by multi-subunit RNA polymerases is achieved through a stepwise process. We show that individual steps contribute differently to discrimination against various erroneous substrates. We define the mechanisms and contributions of each of these steps to the overall fidelity of transcription.

2010-05-07T00:00:00Z Yi, K Mujat, M Park, B H Sun, W Miller, J W Seddon, J M Young, L H de Boer, J F Chen, T C http://hdl.handle.net/2144/3356 2012-01-13T07:00:55Z 2008-12-03T00:00:00Z

Spectral Domain Optical Coherence Tomography for Quantitative Evaluation of Drusen and Associated Structural Changes in Non-Neovascular Age-Related Macular Degeneration Yi, K; Mujat, M; Park, B H; Sun, W; Miller, J W; Seddon, J M; Young, L H; de Boer, J F; Chen, T C BACKGROUND/AIMS:. To demonstrate how spectral domain optical coherence tomography (SDOCT) can better evaluate drusen and associated anatomical changes in eyes with non-neovascular age-related macular degeneration (AMD) compared with time domain optical coherence tomography (TDOCT). METHODS:. Images were obtained from three eyes of three patients with AMD using an experimental SDOCT system. Both a titanium–sapphire (Ti:sapphire) laser and a superluminescent diode (SLD) were used as a broadband light source to achieve cross-sectional images of the retina. A qualitative and quantitative analysis was performed for structural changes associated with non-neovascular AMD. An automated algorithm was developed to analyse drusen area and volume from SDOCT images. TDOCT was performed using the fast macular scan (StratusOCT, Carl Zeiss Meditec, Dublin, California). RESULTS:. SDOCT images can demonstrate structural changes associated with non-neovascular AMD. A new SDOCT algorithm can determine drusen area, drusen volume and proportion of drusen. CONCLUSIONS:. With new algorithms to determine drusen area and volume and its unprecedented simultaneous ultra-high speed ultra-high resolution imaging, SDOCT can improve the evaluation of structural abnormalities in non-neovascular AMD.

2008-12-03T00:00:00Z Bilal, Erhan Rabadan, Raul Alexe, Gabriela Fuku, Noriyuki Ueno, Hitomi Nishigaki, Yutaka Fujita, Yasunori Ito, Masafumi Arai, Yasumichi Hirose, Nobuyoshi Ruckenstein, Andrei Bhanot, Gyan Tanaka, Masashi http://hdl.handle.net/2144/3357 2012-01-13T07:00:55Z 2008-06-11T00:00:00Z

Mitochondrial DNA Haplogroup D4a Is a Marker for Extreme Longevity in Japan Bilal, Erhan; Rabadan, Raul; Alexe, Gabriela; Fuku, Noriyuki; Ueno, Hitomi; Nishigaki, Yutaka; Fujita, Yasunori; Ito, Masafumi; Arai, Yasumichi; Hirose, Nobuyoshi; Ruckenstein, Andrei; Bhanot, Gyan; Tanaka, Masashi We report results from the analysis of complete mitochondrial DNA (mtDNA) sequences from 112 Japanese semi-supercentenarians (aged above 105 years) combined with previously published data from 96 patients in each of three non-disease phenotypes: centenarians (99-105 years of age), healthy non-obese males, obese young males and four disease phenotypes, diabetics with and without angiopathy, and Alzheimer's and Parkinson's disease patients. We analyze the correlation between mitochondrial polymorphisms and the longevity phenotype using two different methods. We first use an exhaustive algorithm to identify all maximal patterns of polymorphisms shared by at least five individuals and define a significance score for enrichment of the patterns in each phenotype relative to healthy normals. Our study confirms the correlations observed in a previous study showing enrichment of a hierarchy of haplogroups in the D clade for longevity. For the extreme longevity phenotype we see a single statistically significant signal: a progressive enrichment of certain "beneficial"patterns in centenarians and semi-supercentenarians in the D4a haplogroup. We then use Principal Component Spectral Analysis of the SNP-SNP Covariance Matrix to compare the measured eigenvalues to a Null distribution of eigenvalues on Gaussian datasets to determine whether the correlations in the data (due to longevity) arises from some property of the mutations themselves or whether they are due to population structure. The conclusion is that the correlations are entirely due to population structure (phylogenetic tree). We find no signal for a functional mtDNA SNP correlated with longevity. The fact that the correlations are from the population structure suggests that hitch-hiking on autosomal events is a possible explanation for the observed correlations.

2008-06-11T00:00:00Z Schapira, Mathilde Buscot, Marie-Jeanne Pollet, Thomas Leterme, Sophie C Seuront, Laurent http://hdl.handle.net/2144/3343 2012-01-12T07:01:56Z 2010-02-24T00:00:00Z

Distribution of Picophytoplankton Communities from Brackish to Hypersaline Waters in a South Australian Coastal Lagoon Schapira, Mathilde; Buscot, Marie-Jeanne; Pollet, Thomas; Leterme, Sophie C; Seuront, Laurent BACKGROUND. Picophytoplankton (i.e. cyanobacteria and pico-eukaryotes) are abundant and ecologically critical components of the autotrophic communities in the pelagic realm. These micro-organisms colonized a variety of extreme environments including high salinity waters. However, the distribution of these organisms along strong salinity gradient has barely been investigated. The abundance and community structure of cyanobacteria and pico-eukaryotes were investigated along a natural continuous salinity gradient (1.8% to 15.5%) using flow cytometry. RESULTS. Highest picophytoplankton abundances were recorded under salinity conditions ranging between 8.0% and 11.0% (1.3 × 106 to 1.4 × 106 cells ml-1). Two populations of picocyanobacteria (likely Synechococcus and Prochlorococcus) and 5 distinct populations of pico-eukaryotes were identified along the salinity gradient. The picophytoplankton cytometric-richness decreased with salinity and the most cytometrically diversified community (4 to 7 populations) was observed in the brackish-marine part of the lagoon (i.e. salinity below 3.5%). One population of pico-eukaryote dominated the community throughout the salinity gradient and was responsible for the bloom observed between 8.0% and 11.0%. Finally only this halotolerant population and Prochlorococcus-like picocyanobacteria were identified in hypersaline waters (i.e. above 14.0%). Salinity was identified as the main factor structuring the distribution of picophytoplankton along the lagoon. However, nutritive conditions, viral lysis and microzooplankton grazing are also suggested as potentially important players in controlling the abundance and diversity of picophytoplankton along the lagoon. CONCLUSIONS. The complex patterns described here represent the first observation of picophytoplankton dynamics along a continuous gradient where salinity increases from 1.8% to 15.5%. This result provides new insight into the distribution of pico-autotrophic organisms along strong salinity gradients and allows for a better understanding of the overall pelagic functioning in saline systems which is critical for the management of these precious and climatically-stress ecosystems.

2010-02-24T00:00:00Z