What is Oxidative Stress

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What is Oxidative Stress?

• Reactive oxygen species
• Superoxide (O2)
• Hydroxyl radical (OH)
• Hydrogen peroxide (H2O2)
• Nitric Oxide (NO)
• Peroxynitrate

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Pho85

• WT Resistant to oxidative stress
• Zwf1D Sensitive to oxidative stress
• Zwf1D/pho85D Resistant to oxidative stress
• Pho85D??CTT1 (catalase) ?suppress
  zwf1D
• ?HSP12 (heat shock protein)
• ?UBI4 (polyubiquitin)

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Preparing A Microarray
1. Cloning Sequencing
Genome
2. Robotic Printing
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Preparing the Samples
WT Yeast
Mutant Yeast
Extraction
RNA
RT reaction
cDNA Labeling
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The Whole Picture
Labeled cDNA
Arrayed Genes
Hybridization
Hybridized Array
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Gene Expression
Wild-Type Yeast Mutant Yeast 1) I
1) 2) II 2) II 3) 3) I
I
I
II II
I
I
I
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Chip Reader
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.tiff data
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Scanalyze/gridding
 
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Genespring
Ratio Mutant Expression / Wildtype expression
Red Mutant gene overexpressed Blue- Mutant gene
underexpressed Grey- No readable
data www.sigenetics.com
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Genome of Yeast PHO 85 knockout
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(green mutant/ red WT)
Three Pho85D Microarrays
(red mutant/green WT)
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Pho85D??CTT1 (catalase) ?suppress
zwf1D ?HSP12 (heat shock
protein) ?UBI4 (polyubiquitin)
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2 Experiments-- Different Results
Red Mutant/ Green WT
Green Mutant/ Red WT
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Cell Cycle control
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Gene FilteringExpression Ratios less then 0.1