Discussion
The goal of this experiment was to identify differentially expressed genes in ΔZMS1 and ΔZMS2 related to oxidative stress. A microarray was used to study the gene expression of ΔZMS1 and ΔZMS2. The RNA was determined to be in the pure range of absorbances (not much protein) and therefore was considered pure. The quantity obtained was also in a good range for microarray use so we were able to obtain at least 8 ug for the labeling procedure. When the RNA was run on a gel, however, it was somewhat degraded (Figure 1). Due to the degradation a different group's RNA was utilized. Because our microarray slide (107) was destroyed during shipping we utilized an array from previous years (Slide 726). Slide 726 had irregular pixal intensity which could have been a result of poor washing technique or incorrectly prepared solutions.
After searching the GCAT database, 29 genes were found related to oxidative stress in yeast. A paired T-test was performed on the 29 genes and found 8 statistically significantly different genes between ΔZMS1 and ΔZMS2 (Table 4). Genes were considered differentially expressed if they passed the t-test with an alpha of 0.01. Of the genes that were differentially expressed between mutants, YBR006W was chosen because of its involvement in human neurological disease. The YBR006W standard name is uga5 and codes for succinate semialdehyde dehydrogenase (SSADH). There was less expression of uga5 in ΔZMS1 compared to ΔZMS2 (Table 4). After further examination of uga5's function, it was found that SSADH is an enzyme in the glutamate catabolic pathway that converts succinate semialdehyde to succinate while reducing NADP (Figure 3)(Coleman 2001). The production of succinate (and therefore reduction of NADP) depends on the presence of gamma aminobutyric acid (GABA) and its enzyme GABA transminase (uga1).
Figure 3. Glutamate Catabolic Pathway. (Coleman 2001)
Previous studies have shown that when increased GABA results in increased SSADH and therefore the reduction of NADP to NADPH (Coleman2001). The reduction of NADPH is an important factor in combating oxidative stress. The uga1 expression ratio was 0.136 in ΔZMS1 and 0.486 in ΔZMS2. This 3-fold difference in upregulation expression levels may have caused differential expression of uga5 in ΔZMS2 when compared to ΔZMS1. ΔZMS2 may be exploiting the glutamate catabolic pathway to combat oxidative stress. ΔZMS1 may be utilizing an alternative pathway, such as the pentose phosphate pathway. Further study should be done to determine what pathway ΔZMS1 may utilize to help combat oxidative stress.