»Home

Role of Transcription Factors in the Response of Saccharomyces cerevisiae to Oxidative Stress

Daniel Robert Rubio; Nathan Day
James Madison University, Harrisonburg, Virginia

Materials & Methods
RNA Isolation from Yeast
Quantification of RNA
Examining RNA Degradation
Preparation of cDNA Probe
Hybridization of cDNA Probe
Microarray Analysis

Results
RNA Isolation from Yeast
Gridding and Segmenting
Transformation of Microarray Data
Microarray Analysis

Discussion
Yeast Growth Phase Analysis
RNA Quality and Quantity
Microarray Analysis
Genes of Interest

Literature Cited

Picture taken from http://www.wormbook.org/chapters/www_germlinegenomics/germlinegenomicsfig1.jpg.

Abstract.  The growing science of microarray technology is allowing scientists a new way to examine gene expression at the organismal level. This technology utilizes the hybridization of dye-labeled probes isolated from a specific organism or variable, such as disease, to the genome of the organism being studied.  In this experiment, microarray technology was utilized in order to determine the role transcription factors had on the oxidative stress response of  Saccharomyces cerevisiae (yeast) through the study of knock-out mutants ∆ZMS1 and ∆ZMS1/∆ZMS2.  It was hypothesized that genes involved in the oxidative stress response would be down-regulated in the knock-out mutants, because the mutants grow poorly in the presence of reactive oxygen species.  The study of this eukaryotic model organism would hopefully shine light on how human somatic cells respond to oxidative stress.  To test our hypothesis, dye-labeled cDNA was constructed through reverse transcription of isolated RNA from the different yeast cultures.  These labeled cDNA probes were then hybridized to microarray slides containing the yeast genome.  Using microarray analysis software, the data was collected from the slide and analyzed for genes with significant over-expression or under-expression.  The slides used in microarray analysis were TR104_w595, TR104_w685, TR106_w595 and TR106_w685, with the green channel (_w595) being the wild-type yeast, and the red channel (_w685) being the knock-out mutants.  Twelve genes were identified as significantly over-/under-expressed in the yeast knock-out mutants.  Two of the six genes determined to be down regulated are known to be involved in the cell's stress response (YFL014W and YBR072W) and the remaining are of unknown function. This information indicates that these four genes may be involved in oxidative stress response and we suggest further investigation into their role in the cell.