investigation of microarray data from douillard’s heliocobacter pylori experiment samantha hurndon...
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![Page 1: Investigation of Microarray Data from Douillard’s Heliocobacter pylori Experiment Samantha Hurndon Isaiah Castañeda](https://reader036.vdocuments.net/reader036/viewer/2022072006/56649cf85503460f949c999a/html5/thumbnails/1.jpg)
Investigation of Microarray Data from Douillard’s Heliocobacter
pylori Experiment
Samantha Hurndon
Isaiah Castañeda
![Page 2: Investigation of Microarray Data from Douillard’s Heliocobacter pylori Experiment Samantha Hurndon Isaiah Castañeda](https://reader036.vdocuments.net/reader036/viewer/2022072006/56649cf85503460f949c999a/html5/thumbnails/2.jpg)
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
![Page 3: Investigation of Microarray Data from Douillard’s Heliocobacter pylori Experiment Samantha Hurndon Isaiah Castañeda](https://reader036.vdocuments.net/reader036/viewer/2022072006/56649cf85503460f949c999a/html5/thumbnails/3.jpg)
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
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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
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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
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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
![Page 7: Investigation of Microarray Data from Douillard’s Heliocobacter pylori Experiment Samantha Hurndon Isaiah Castañeda](https://reader036.vdocuments.net/reader036/viewer/2022072006/56649cf85503460f949c999a/html5/thumbnails/7.jpg)
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
![Page 8: Investigation of Microarray Data from Douillard’s Heliocobacter pylori Experiment Samantha Hurndon Isaiah Castañeda](https://reader036.vdocuments.net/reader036/viewer/2022072006/56649cf85503460f949c999a/html5/thumbnails/8.jpg)
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
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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
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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
![Page 11: Investigation of Microarray Data from Douillard’s Heliocobacter pylori Experiment Samantha Hurndon Isaiah Castañeda](https://reader036.vdocuments.net/reader036/viewer/2022072006/56649cf85503460f949c999a/html5/thumbnails/11.jpg)
GenMAPP was used to find significant increases in gene expression
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Using GenMAPP, a list of the most significantly suppressed genes was
compiled
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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
![Page 14: Investigation of Microarray Data from Douillard’s Heliocobacter pylori Experiment Samantha Hurndon Isaiah Castañeda](https://reader036.vdocuments.net/reader036/viewer/2022072006/56649cf85503460f949c999a/html5/thumbnails/14.jpg)
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
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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
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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
![Page 17: Investigation of Microarray Data from Douillard’s Heliocobacter pylori Experiment Samantha Hurndon Isaiah Castañeda](https://reader036.vdocuments.net/reader036/viewer/2022072006/56649cf85503460f949c999a/html5/thumbnails/17.jpg)
None of the most significantly changed genes were RpoN-dependent genes
• Our
•Perhaps there was more significant transcriptional activity than Class II genes
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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