PMC:3091627 / 25378-33251 JSONTXT

{"project":"2_test","denotations":[{"id":"20537153-11866264-10781747","span":{"begin":304,"end":306},"obj":"11866264"},{"id":"20537153-15194637-10781748","span":{"begin":378,"end":380},"obj":"15194637"},{"id":"20537153-15504269-10781749","span":{"begin":976,"end":978},"obj":"15504269"},{"id":"20537153-15504269-10781750","span":{"begin":1503,"end":1505},"obj":"15504269"},{"id":"20537153-15504269-10781751","span":{"begin":2285,"end":2287},"obj":"15504269"},{"id":"20537153-15504269-10781752","span":{"begin":3162,"end":3164},"obj":"15504269"},{"id":"20537153-9521922-10781753","span":{"begin":5404,"end":5406},"obj":"9521922"},{"id":"20537153-9521923-10781754","span":{"begin":5650,"end":5652},"obj":"9521923"},{"id":"20537153-11282977-10781755","span":{"begin":5755,"end":5757},"obj":"11282977"},{"id":"20537153-12824403-10781756","span":{"begin":6953,"end":6955},"obj":"12824403"},{"id":"20537153-16407324-10781757","span":{"begin":7077,"end":7079},"obj":"16407324"}],"text":"Methods\n\nH. pylori strains, and growth\nWe examined 120 H. pylori strains isolated from patients from different areas of France enrolled in 3 multi-center studies carried out by 1) the Groupe d'Etude Français des Helicobacter (G.E.F.H.), 2) the Groupe d'Etude Français des Lymphomes Digestifs (G.E.L.D.) [37] and of the Fédération Française de Cancérologie Digestive (F.F.C.D.) [38], and 3) the Groupe d'Etude des Lymphomes de l'Adulte (G.E.L.A.). Criteria for patient inclusion were age (\u003e55 years), suffering from chronic gastritis (n = 33), duodenal ulcer without intestinal metaplasia (27), intestinal metaplasia without ulcer (n = 17). We identified 43 strains from patients with gastric MALT lymphoma. H. pylori was isolated from one biopsy specimen following biopsy homogenization and culture under microaerophilic conditions (5-6% 02, 8-10% CO2, 80-85% N2) on blood agar medium (BA; Oxoid blood agar base N°2) supplemented with 10% horse blood, as reported previously [39]. One colony was selected at random from each primary culture; it was then sub-cultured and used to prepare chromosomal DNA. This DNA was extracted from 48-hour-old confluent cells using the QIAamp Tissue kit (Qiagen, Chatsworth, CA) according to the manufacturer's recommendations.\n\nIn house DNA macroarray membrane preparation\nA total of 254 PCR products were amplified in four 96-well microtiter plates, corresponding to 41 ubiquitous and 213 non-ubiquitous genes from the genome of strain 26695 as previously described [39]. Briefly, amplification reactions were performed in 2 × 100 μl reaction volumes, in which 2 μl of DNA corresponding to the recombinant plasmid containing the full-length CDS (CoDing Sequence) inserted into the pILL570-derivative vector was used as template. Each PCR product was sequenced to confirm the identity of the gene, and was then spotted in triplicate onto a nylon membrane (Qfilter, Genetix 22.2 × 22.2 cm, N+) using a Qpix robot (Genetix). Denaturated 26695 genomic DNA was spotted in triplicate at the four corners of the membrane (positive controls) and seven squares were left empty as negative controls. Following spot deposition, membranes were fixed for 15 minutes in 0.5 M NaOH 1.5 M NaCl, washed briefly in distilled water, and stored wet at -20°C until use [39].\nAliquots of 250 μl of DNA were labeled by random priming with 2 μl of 33P-dCTP. Labeling was performed for 3 hours at room temperature. Unincorporated radionucleotides were removed by purification on Quick Spin Sephadex G-25 columns (Roche Diagnostics). Immediately before being used for hybridization experiments, the sonicated, labeled, and purified chromosomal DNA was heat-denaturated and cooled on ice. Hybridization was conducted in 5 ml prewarmed (65°C) hybridization mixtures containing the heat denaturated probe, with overnight incubation. Membranes were then washed and exposed for 25 hours to a phosphoimager screen (Molecular Dynamics).\nScreens were scanned on a Storm 860 machine (Molecular Dynamics). Image analysis and quantification of hybridization intensities for each spot were performed using the Xdots Reader program (COSE) and determined in pixels [39]. The intensity of the background surrounding each spot was substracted from that of each of the spots. Twenty-one homologous hybridizations were performed. The average intensity of the 41 ubiquitous genes was calculated for each reference array. This number served to allocate a reference array to each heterologous hybridization (average of the ubiquitous spots from the heterologous and the homologous reference hybridizations were not significantly different, Student's test), to calculate the ratio used for normalization. Following normalization, the data were analyzed by attributing a binary score (presence/absence - Additional file 1) or by multidimensional analysis based on continuous intensity values (Figure 1 and Figure 2). To define the cutoff ratio for the presence/absence of a gene, we analyzed the results for the sequenced H. pylori J99 DNA hybridized with H. pylori 26695; the threshold for the presence of a gene was defined as \u003e0.25. The multidimensional analyses (Genesis software) for the hierarchical clustering as well as for the Principal Component Analysis were performed using the 254 continuous values from the 120 heterologous hybridization experiments, each corresponding to (log10normalized intensity values of strain 26695) minus (log10normalized intensity values of the heterologous strain) (i.e. log26695-logheterol.strain).\n\nSequencing and annotation of the B38 genome\nGenomic DNA was randomly sheared by nebulization (HydroShear, GeneMachines) and the ends were enzymatically repaired. SmaI fragments (1.5-4 kb) were inserted into plasmid vector pBAM3/SmaI (derived from pBluescript KS and constructed by R. Heilig). Large (35-45 kb) DNA fragments generated from partial BamHI-restriction were inserted into the cosmid vector pHC79/BamHI.\nPlasmid DNA was prepared with the TempliPhi DNA sequencing template amplification kit (GE Healthcare-Bio-Sciences). Cosmid DNA was purified with the Montage BAC Miniprep96 Kit (Millipore). Sequencing reactions were performed from both ends of DNA templates using ABI PRISM BigDye Terminator cycle sequencing ready reactions kits and were run on a 3700 or a 3730 xl Genetic Analyzer (Applied Biosystems).\nSequence data base calling was carried out using Phred [40]. Sequences not meeting our production quality criteria (at least 100 bases called with a quality over 20) were discarded. Sequences were screened against plasmid vector and E. coli sequences. The traces were assembled using Phrap and Consed [41]. Whole genome shotgun sequencing was performed to ensure approximately 11-fold coverage. Autofinish [42] was used to design primers for improving regions of low quality sequence and for primer walking along templates spanning the gaps between contigs. Several strategies were used to orientate contigs and to enable directed PCR-based approaches to span the gaps between contigs. These strategies included linking isolates and a Blast-based approach, which identified contigs with hits to the H. pylori strain 26695 genome. Various combined PCR techniques were used to amplify genomic or cosmid DNA, to close the gaps between the final contigs. Outward-directed primers were designed for each of the contig ends; the primer sequences were subsequently checked and confirmed to be unique to the genome. This combined PCR process required approximately 200 PCR reactions pairing each of the primers. In addition, two cosmid isolates containing a rDNA operon copy each, were completely sequenced by sub-cloning into a pSMART-LC vector (Lucigen Corp.). The error rate was less than 1 error per 10,000 bp in the final assembly. The complete genome sequence was obtained from 40 153 sequences, resulting in 14-fold coverage.\nAMIGene software was used to predict which CDSs were likely to encode proteins [43]. The set of predicted genes underwent automatic functional annotation using the set of tools listed in Vallenet et al. [28]. All these data (syntactic and functional annotations, results of comparative analysis) are stored in a relational database, called PyloriScope. Manual validation of the automatic annotation was performed using the MaGe (Magnifying Genomes, http://www.genoscope.cns.fr) web-based interface, which allows graphic visualization of the annotations enhanced by the synchronized representation of synteny groups in other genomes chosen for comparison.\n\nAccession Numbers\nThe EMBL Nucleotide Sequence Database http://www.ebi.ac.uk/embl accession number for the H. pylori strain B38 chromosome is [EMBL:FM991728].\nAll data and comparative genomics concerning the H. pylori B38 genome are stored in PyloriScope http://www.genoscope.cns.fr/agc/mage, a related database that is available to the public."}