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Investigation of Microarray Data from Douillard’s Heliocobacter pylori Experiment

Investigation of Microarray Data from Douillard’s Heliocobacter pylori Experiment. Samantha Hurndon Isaiah Castañeda. Outline. Francois Douillard and his colleagues published a paper in 2009 about the Flik gene of H. pylori

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Investigation of Microarray Data from Douillard’s Heliocobacter pylori Experiment

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  1. Investigation of Microarray Data from Douillard’s Heliocobacter pylori Experiment Samantha Hurndon Isaiah Castañeda

  2. Outline • Francois Douillard and his colleagues published a paper in 2009 about the Flik gene of H. pylori • Statistical data was gathered to determine which genes in this experiment underwent the greatest changes • MAPPFinder was used to determine the most upregulated and downregulated genes • Data indicated that many of the top 10 most significant genes dealt with transcription • Data shows that RpoN dependent genes were not among the most significantly changed

  3. Motility is a key feature of H. pylori and is required for colonization and persistence • There are three classes (stages) of regulation • Class I: transcription of the early flagellar genes • Class II: RpoN regulation of the middle flagellar structural genes. (fliKencodes hook-length-control pro) • Class III: FliA and an anti sigma factor (FlgM) • FliK is the gene that controls hook length during flagellar assembly

  4. CCUG17874 & NCTC26695 Strains were used for the microarray dataset • The wild type CCUG17874 DNA was labeled with dCTP Cy3 • Cy3-dCTP is a fluorescent cyan dye • Wild type NCTC26695 DNA was labeled with dCTP Cy5 • Cy5-dCTP is a fluorescent red dye

  5. Microarray chips were hybridized • Cy5 hp0906KO (C1) with Cy3 CCUG17874 (A3) → Hyb:32525 • Cy3 CCUG17874 (A3)-23856 with Cy5 CCUG17874 (C1) → Hyb:32519 • Cy5 hp0906KO (B1) with Cy3 CCUG17874 (A2) → Hyb:32524 • Cy3 CCUG17874 (A1) with Cy5 hp0906KO (A1) → Hyb:32523 • Cy5 CCUG17874 (B1) with Cy3 NCTC26695 (B1) → Hyb:32528 • Cy3 CCUG17874 (A5) with Cy5 hp0958 KO (B1) → Hyb:32521 • Cy3 NCTC26695 (A1) with Cy5 CCUG17874 (A1) → Hyb:32526 • Cy5 hp0958 KO (A1) with Cy3 CCUG17874 (A4) → Hyb:32520 • Cy3 CCUG17874 (A2)-23855 with Cy5 CCUG17874 (B2)-23817 → Hyb:35218 • Cy3 CCUG17874 (A1)-23769 with Cy5 CCUG17874 (A1)-23814 → Hyb:32517 • Cy3 CCUG17874 (A6) with Cy5 hp0958 KO (C1) → Hyb:32522

  6. FliK protein is required to turn off the RpoN regulation during flagellar assembly • Under the control of the RpoN there is an increase in transcription of genes • Gene expression has been significantly changed after mutation on fliK • Flik mutation affected the transcription of almost all class II genes in flagellar regulon

  7. Outline Francois Douillard and his colleagues published a paper in 2009 about the Flik gene of H. pylori Statistical data was gathered to determine which genes in this experiment underwent the greatest changes MAPPFinder was used to determine the most upregulated and downregulated genes Data indicated that many of the top 10 most significant genes dealt with transcription Data shows that RpoN dependent genes were not among the most significantly changed

  8. Statistical analysis was performed on the microarray dataset • Data was gathered from the European Bioinformatics Institute website • There were 3 experiments • hp0958KO • hp0906KO • CCUG17874 • Averages, Tsats, and P-values were calculated for every gene of each experiment

  9. HP0906 KO had the highest amount significantly changed genes

  10. Outline • Francois Douillard and his colleagues published a paper in 2009 about the Flik gene of H. pylori • Statistical data was gathered to determine which genes in this experiment underwent the greatest changes • MAPPFinder was used to determine the most upregulated and downregulated genes • Data indicated that many of the top 10 most significant genes dealt with transcription • Data shows that RpoN dependent genes were not among the most significantly changed

  11. GenMAPP was used to find significant increases in gene expression

  12. Using GenMAPP, a list of the most significantly suppressed genes was compiled

  13. Outline Francois Douillard and his colleagues published a paper in 2009 about the Flik gene of H. pylori Statistical data was gathered to determine which genes in this experiment underwent the greatest changes MAPPFinder was used to determine the most upregulated and downregulated genes Data indicated that many of the top 10 most significant genes dealt with transcription Data shows that RpoN dependent genes were not among the most significantly changed

  14. Many of the most significant genes deal with transcription • HP1555 – Elongation FactorAssociates with the EF-Tu.GDP complex and induces the exchange of GDP to GTP. It remains bound to the aminoacyl-tRNA.EF-Tu.GTP complex up to the GTP hydrolysis stage on the ribosome • HP1557 - Flagellar hook-basal body protein FliE • HP0912 – Predicted to be an outer membrane protein • HP1203 - Transcription antitermination protein • HP0601- Flagellin A Part of the Flagellar assembly and affects motility • HP1429 – Predicted to be a polysialic acid capsule expression protein • HP0962 - Acyl carrier protein • HP1427 – Predicted to be a histidine and glutamine-rich metal binding protein • HP1210 - Serine acetyltransferase • HP0264 – Predicted to be an ATP-dependent chaperone protein ClpB

  15. Douillard found that Flik mutation affected the transcription of almost all class II genes in flagellar regulon • Shown above are the differential expression ratios of all known • Flagellar genes in the fliK mutant relative to the wild-type

  16. Outline • Francois Douillard and his colleagues published a paper in 2009 about the Flik gene of H. pylori • Statistical data was gathered to determine which genes in this experiment underwent the greatest changes • MAPPFinder was used to determine the most upregulated and downregulated genes • Data indicated that many of the top 10 most significant genes dealt with transcription • Data shows that RpoN dependent genes were not among the most significantly changed

  17. None of the most significantly changed genes were RpoN-dependent genes • Our • Perhaps there was more significant transcriptional activity than • Class II genes

  18. References • Douillard, Francois, Ryan Kieran, Jason Hinds, and Paul O'Toole. "Effect of FliK mutation on the transcriptional activity of the sigma factor RpoN in Heliobacter pylori." Microbiology. 21.4 (2009): 1901-1911. • http://www.uniprot.org/uniprot/P55975 • http://www.genome.jp/dbget-bin/www_bget?hpy:HP1557 • http://combrex.bu.edu/DAI?command=SciBay&fun=gene&geneID=899441 • http://www.genome.jp/dbget-bin/www_bget?hpy+HP1203 • http://www.genome.jp/dbget-bin/www_bget?hpy:HP0601 • http://combrex.bu.edu/DAI?command=SciBay&fun=gene&geneID=899866 • http://www.gene-profiles.org/gene/hp0962-helicobacter-pylori-26695-899495 • http://combrex.bu.edu/DAI?command=SciBay&fun=gene&geneID=899868 • http://www.genome.jp/dbget-bin/www_bget?hpy:HP1210 • http://combrex.bu.edu/DAI?command=SciBay&fun=gene&geneID=899187

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