Simple cDNA normalization using kamchatka crab duplex-specific nuclease

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Simple cDNA normalization using kamchatka crab duplex-specific nuclease

Oxford University Press
Publication Date
Jan 01, 2004
  • Biology


OP-NARE131201 1..18 The complex methylome of the human gastric pathogen Helicobacter pylori Juliane Krebes1,2, Richard D. Morgan3, Boyke Bunk2,4, Cathrin Spro¨er2,4, Khai Luong5, Raphael Parusel1, Brian P. Anton3, Christoph Ko¨nig5, Christine Josenhans1,2, Jo¨rg Overmann2,4, Richard J. Roberts3, Jonas Korlach5 and Sebastian Suerbaum1,2,* 1Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Carl-Neuberg-Straße 1, 30625 Hannover, Germany, 2German Center for Infection Research, Hannover-Braunschweig Site, Carl- Neuberg-Straße 1, 30625 Hannover, Germany, 3New England Biolabs, 240 County Road, Ipswich, MA 01938, USA, 4Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, 38124 Braunschweig, Germany and 5Pacific Biosciences, 1380 Willow Road, Menlo Park, CA 94025, USA Received September 5, 2013; Revised November 1, 2013; Accepted November 4, 2013 ABSTRACT The genome of Helicobacter pylori is remarkable for its large number of restriction-modification (R-M) systems, and strain-specific diversity in R-M systems has been suggested to limit natural trans- formation, the major driving force of genetic diver- sification in H. pylori. We have determined the comprehensive methylomes of two H. pylori strains at single base resolution, using Single Molecule Real-Time (SMRT�) sequencing. For strains 26695 and J99-R3, 17 and 22 methylated sequence motifs were identified, respectively. For most motifs, almost all sites occurring in the genome were detected as methylated. Twelve novel methylation patterns corresponding to nine recognition sequences were detected (26695, 3; J99-R3, 6). Functional inactivation, correction of frameshifts as well as cloning and expression of candidate methyltransferases (MTases) permitted not only the functional characterization of multiple, yet undescribed, MTases, but also revealed novel features of both Type I and Type II R-M systems, including frameshift-mediated changes of sequence specificit

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