統合TV 統合TVは、ライフサイエンス統合データベースセンター(DBCLS)が発信する生命科学分野の有用なデータベース(DB)やウェブツールの活用法を動画で紹介するウェブサイトです。くわしくは はじめての方へ をご覧ください。ご質問、ご要望は、お問い合わせ もしくは ライフサイエンスQA からお願いします。TogoTV beta ('togo' means 'integration' in Japanese) is an archive of tutorial videos expounding how to use biological databases and tools. Although most of contents are currently depicted in Japanese, you can find English contents in TogoTV international. Please post any comments, suggestions, and requests at each content page, e-mail to the address described at the bottom of each page or contact form. This site is provided by Database Center for Life Science (DBCLS) and funded by MEXT Integrated Database Project in Japan.

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2011-12-14

_ [presentation][English] ゲノムメチル化とエピジェネティクスII:Genome Methylation and Epigenetics II: Helicobacter pylori

ゲノムメチル化とエピジェネティクスII: Helicobacter pylori

本日の統合TVは、2011年度夏学期(4〜7月)に開講された、東京大学グローバルCOEプログラム「ゲノム情報ビッグバンから読み解く生命圏新領域創成科学特別講義IIから、Department of Biochemistry, Indian Institute of Science の Prof. Desirazu Narasimha Rao による「ゲノムメチル化とエピジェネティクスII: Helicobacter pylori」をお送りします。

Youtube版はこちらです。

This lecture is talked by Prof. Desirazu Narasimha Rao from Department of Biochemistry, Indian Institute of Science entitled "Genome Methylation and Epigenetics II: Helicobacter pylori". It is a part of "Frontier Sciences Special Lecture Series II" in The University of Tokyo Global COE Program: Deciphering Biosphere from Genome Big Bang.

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Keywords: GCOE,Genome big bang, Helicobacter pylori

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Helicobacter Pylori Number of Restriction-Modification systems in different bacteria Restriction-Modification (R-M) systems in Helicobacter pylori Helicobacter pylori Evolution during disease progression Chromosomal location of hp0050 and hp0051 HP00051 protein sequence Modeled Structure of HP0051 Purification of hp0051 from genomic DNA of H. pylori strain 26695 Oligomeric status Kinetic and Catalytic properties of HP0051 C5 Cytosine Methyltransferase Kinetic parameters of HP0051 catalyzed methylation of DNA Metal binding motif is in close proximity with catalytic motif Effect of metal on HP0051 activity Comparison on between different HP0051 isoforms Over expression of HP0051 induces random mutations in E. coli strain BL21(DE3) MALDI-MS spectra of trypsin digested HP0051 from Helicobacter pylori MALDI-MS spectra of trypsin digested HP0051 isoforms from Helicobacter pylori Kinetic parameters of HP0051 (in vitro translated) catalyzed methylation of DNA In vitro methylation assay Sensitivity to Restriction Endonuclease Microarray Analysis Effect of HP0051 overexpression on Mismatch Repair genes dam is required for strand discrimination in Mismatch repair pathway Distribution of HP0051 cognate sequence in promoter region of MMR pathway genes RT PCR analysis Effect of expression of M.HpyAVIAB on Dam activity in E. coli BL21(DE3) Effect of expression of M.HpyAVIAS on transcript levels of genes involved in MMR and SOS pathway Effect of methylation by M.HpyAVIB on ssb promoter strength In vivo screen for determination of mutation frequency PCR amplification of hp0051 from clinical isolates Sequence analysis of HP0051 from clinical isolates hp0051 shows phase variation in vivo Distribution of hp0051 in symptomatic (Kolkata) strains hp0051 knockout construct hp0051 - chloramphenicol knockout construct Transformation of H. pylori hp0051 knockout construct Screening for H. pylori strain 26695 and SS1 hp0051 knockout Characterization of H. pylori strain 26695 hp0051 deletion mutant H. pylori-stimulated interleukin-8 (IL-8) promotes cell proliferation in infected gastric mucosa IL-8 Assay Assessment of IL-8 production by AGS cell line Motility Assay Microarray analysis of H. pylori wild type vs hp0051 deletion mutant hp0051 deletion affects the expression of genes involved in different pathway Outer Membrane Proteins Deletion of hp0051 results in up regulation of OMP LPS diversity within an H. pylori community is important for a persistent human infection Lipopolysaccharide Diversity Evolving in Helicobacter pylori Communities through Genetic Modifications in Fucosyltransferases Molecular mechanism of action of major Helicobacter pylori virulence factors Deletion of hp0051 results in up regulation of virulent factors Survival of a pathogen in host depends upon its ability to modulate the balance between virulence and avirulence SUMMARY Chromosomal location of hp0050 and hp0051 HP0050 protein sequence PCR amplification, over expression and purification of H. pylori HP0050 MTase Peptide finger map of HP0050 protein Characterization of AdoMet binding motif (F195S) and catalytic motif (Y32L) HP0050 mutant proteins Specificity of HP0050 MTase HP0050 methylates two adjacent residues on the same strand HP0050 methylates both adenines in 5 GAAG 3 HP0050 methylates DNA in a non processive manner HP0050 methylates both adenines in 5 GAAG 3 in a non processive manner Methylation activity of HP0050 MTase homologs from H. pylori strains 26695, PG227 and 128 Characterization of hp0050 deletion mutant derivative of H. pylori Insertion of a single nucleotide can bring both ORFs in frame and </p>
<p>codes for a single protein PCQ and DPPY are the catalytic domain and FCGAG and FMGSG are AdoMet binding  domain of HP0051 and HP0050 respectively Single mRNA codes HP0051 and HP0050 in vitro translation of HP0051_HP0050 Fusion protein Phase variable genes in Helicobacter pylori Based on the relative arrangements of TRD, motif I and motif IV methyltransferases are characterized in six groups Region View: hp1369_hp1370 Addition of a nucleotide to the poly G stretch of hp1369 forms a functional epsilon type of Methyltransferase hp1369_hp1370 shows a deletion of 300-600 bp in most of the H. pylori strains PCR amplification of hp1369_hp1370 from H. pylori strain 26695 Breaking the -G- track hp1369_1370 shows phase variation HP1369_1370 fusion MTase recognizes and methylates 5 TCAGC 3 hp0592-hp0593 Operon Amino Acid Sequence of HP0593 Identification of HP0593 Methylation assay with Unmethylated vs Methylated duplex DNA Determination of HP0593 MTase activity Distribution of hp0050 cognate sequence in the promoter sequences of H. pylori Expression of hp0593 in response to acidic stress Confirmation of hp0593 knockout construct Growth kinetics of wild-type and knock-out strain Microarray analysis of knock-out and wild-type strain Differential expression of genes Analysis of transcript levels using RT-PCR Cell morphology study Transmission electron microscope analysis Downregulated genes hp1366-hp1367-hp1368: A functional Type IIS R-M System Purification of HP1367 (GST Tag) and HP1368 (HIS Tag) HP1367 is an N6 adenine and HP1368 is a N4 cytosine methyltransferase HP1367 methylates upper strand and HP1368 methylates lower strand of sequence 5 GAAGA 3 Restriction-Modification systems in H. pylori is dynamic and evolving Tensions between genomic integrity and diversification in H. pylori Evolution of Methyltransferase with new or relaxed specificity and functions beyond genome protection Acknowledgments Acknowledgments


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