Transcript Douillard found that Flik

Investigation of Microarray Data
from Douillard’s H. 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
• GenMAPP was used to determine the most upregulated
and downregulated genes
• MAPPFinder 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
Eleven microarray chips were
hybridized
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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
• GenMAPP was used to determine the most upregulated
and downregulated genes
• MAPPFinder 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
• GenMAPP was used to determine the most upregulated
and downregulated genes
• MAPPFinder 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
• GenMAPP was used to determine the most upregulated
and downregulated genes
• MAPPFinder 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
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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
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HP1557 - Flagellar hook-basal body protein FliE
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HP0912 – Predicted to be an outer membrane protein
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HP1203 - Transcription antitermination protein
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HP0601- Flagellin A
Part of the Flagellar assembly and affects motility
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HP1429 – Predicted to be a polysialic acid capsule expression protein
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HP0962 - Acyl carrier protein
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HP1427 – Predicted to be a histidine and glutamine-rich metal binding protein
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HP1210 - Serine acetyltransferase
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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
• GenMAPP was used to determine the most upregulated
and downregulated genes
• MAPPFinder 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
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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.
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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
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