Abstract       Introduction        Materials and Methods       Results      Discussion      Sources Cited

Oxidative stress is a contributing factor to human diseases like neurodegenerative diseases, cancer, and cardiovascular disease (Professor Kim Slekar Lecture, March 2009) as well as senescence associated with nuclear and mitochondrial DNA damage (Zglinicki, 2003). Reactive oxygen species (ROS) like, hydroxyl radical, superoxide anion, and hydrogen peroxide are natural byproducts of normal metabolic processes, approximately 2% of the electrons which pass through the electron transport chain result in ROS (Zglinicki, 2003). To counter ROS all cells have evolved enzymes and can make other small molecules which neutralize the damaging molecules. The pentose phosphate pathway is a biosynthetic pathway which makes five carbon ribose sugars for nucleotide synthesis and is a source of NADPH, a reducing agent, which is able to combat oxidative stress (Professor Kim Slekar Lecture, March 2009).  To study oxidative stress Dr. Slekar, a James Madison University geneticist, mutated, in Saccharomyces cerevisiae, the ZMS1 and ZMS2 genes which are multicopy supressors of ZWF1. The ZWF1 gene codes for Glucose-6-Phosphate that plays a major role on the pentos phosphate pathway which makes NADPH (Slekar et. al., 1996).  When the ZMS1 or ZMS2 genes are knocked out the yeast cells have a greater ability to combat oxidative stress (Professor Kim Slekar Lecture, March 2009).

        To create a micro array are usually polymerase chain reaction (PCR) products which were created using cDNA libraries. These cDNAs are printed onto glass slides as spots 100–300 um in size and are spaced apart on a grid (Figure 1).  Arrays containing more than 30,000 cDNAs can be created on a normal microscope slide.  Labeled mRNA from cells are then extracted converted to DNA and labeled using different fluorescent dyes such as Cy3 (green) and Cy5 (red).  This allows mRNAs from two different cell populations to be labeled in different colors, mixed and hybridized simultaneously resulting in competitive binding of these two cell types.  A gene which is up regulated will hybridize more quickly and the majority of that spot will have its labeled DNA on it.  Intensity readings of each spot can now be read and a ratio of red to green dye is used for analysis which gives a relative transcript level of the two different cells.

Figure1: cDNA microarrays. Array preparation: inserts from cDNA libraries are amplified using PCR and the products are printed onto glass.  Target preparation: RNA from two different cell types is used to synthesize single-stranded cDNA and is labeled with two different fluorescent dyes.  Both samples are mixed and hybridized to the array surface which results in competitive binding of the two different labeled cDNAs to the corresponding array elements. High-resolution fluorescence scanning of the array using two different wavelengths corresponding to the dyes gives a relative intensities and ratios of the intensities for the genes represented on the array.

A local approach to analyzing microarrays looks at up and down regulated genes between two different cells (Figure 2).  The genes are compared against a gene list, and up or down regulated genes can be used for further studies.

Figure 2:  The local approach for analyzing microarray data uses just two different conditions to determine up or down regulated genes.  These are compared with a list of known genes and up or down regulated genes can be used for further studies.

In this experiment ZMS2 and ZMS1 mutants were studied using microarray slides. The global gene expression of the mutants was compared to wild type yeast to determine what genes were up or down regulated by the removal of the ZMS genes. Ideally, this data could lead to new avenues of research for Dr. Slekar who is trying to find ways to combat oxidative stress.