Discussion
The objective of this experiment was to identify genes that were expressed differently between wildtype yeast (Saccharomyces cerevisiae) and ∆ZMS1 and ∆ZMS2 mutation strains. The mutations were thought to be linked to oxidative stress. Initially, RNA was extracted from three different strains, including Wildtype, ∆ZMS1 and ∆ZMS2. The data collected from this step can be found in Table 1 on the results page. These strains were then tested for purity via a nanodrop, comparing the absorbance at 260nm/280nm. The data is found in Table 2 of the results section. The yeast obtained and tested were found to have an acceptable purity ratio as shown in the A260/A280 reading. The concentration of the RNA at this step was slightly lower than desired. However, this could have been due to the degradation of the RNA, although this is unlikely. Although, a lower concentration was obtained than desired, the obtained concentration was still able to be analyzed via gel electrophoresis. The cDNA, created from our RNA samples, was then tagged and added to a microarray slide. The slides were sent off for analysis at Davidson College. Unfortunately, in transit the slides were broken. Due to this event, a slide from previous year’s experiment was used for further analysis. A sample of the microarray data from the slide used is also found on the results page as Figure 2. Overall, the BIO480 class microarray data was insufficient for analysis. This could be due to possible error during the concentration of cDNA using the micro concentrator. Other sources of error could have arisen due to errors in the hybridization procedure.
After inputting the data into Magic Tool, we analyzed eighteen genes related to glycogenesis. Glycogenesis is the conversion of glucose to glycogen. This occurs when the yeast does not have a lot of ATP or during a rest period in the cell. During a period where the yeast has a sufficient supply of ATP the glycogenesis process is inhibited because the yeast does not have a need to store energy (elmhurst.edu). Glycogenesis was selected as a potentially interesting area of analysis due to the general trend of differential gene expression in ∆ZMS1 and ∆ZMS2 mutation strains compared to the wildtype strain. Glycogenesis can be related to oxidative stress in that the reduction and oxidation of polysaccharides can produce ROS and superoxides. Eight genes were found to be upregulated by at least two-fold and two genes downregulated by at least two-fold. It appears that the majority of these glycogenesis-related genes are upregulated in the mutant strains, from the wildtype yeast strain. However, the cellular effects of upregulation/downregulation of glycogenesis-related genes is unknown. This data is represented by Table 3 and Figure 3. Future experiments include RT-PCR analysis of some of these genes to accurately determine expression levels. A DNA microarray analysis of ZMS1 and ZMS2 double knockout yeast compared to wildtype yeast could provide valuable information as well.
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