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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 • 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 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. (fliK encodes hook-length-control pro) – Class III: FliA and an anti sigma factor (FlgM) • FliK is the gene that controls hook length during flagellar assembly 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 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 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 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 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 HP0906 KO had the highest amount significantly changed genes Gene P-Values HP0958 KO HP0906 KO CCUG17874 <0.05 187 647 298 <0.01 36 164 53 <0.001 3 12 9 <0.0001 0 1 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 GenMAPP was used to find significant increases in gene expression Using GenMAPP, a list of the most significantly suppressed genes was compiled 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 Many of the most significant genes deal with transcription • HP1555 – Elongation Factor Associates 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 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 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 None of the most significantly changed genes were RpoN-dependent genes • Our •Perhaps there was more significant transcriptional activity than Class II genes 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-26695899495 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 • • •