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Oxidative stress is caused by an imbalance between the production of reactive oxygen and a biological system's ability to detoxify the reactive oxygen molecules produced. This can cause cancer, neurodegenerative disease, and cardiovascular disease. There are anti-oxidants which can inhibit the functions of the reactive oxygen molecules reducing oxidative stress. Yeast carries a gene zwf1∆ which codes for Glucose-6 phosphate dehydrogenase which acts as anti-oxidant. Previous studies have found when zwf1∆ is knocked out high levels of zms1∆ and zms2∆ suppress the phenotypes of the knockout mutant (Slekar 2008). This study used microarray analysis to compare zms2∆ and wild-type (WT).
A total of seven genes were up or down regulated in the zms2Δ mutant when compared to the wild type. Genes found to be down regulated were SPR28 (locus tag YDR218C) and MRPL40 (locus tag YPL173W). The MRPL40 gene produces a protein that functions as the large subunit of the yeast mitochondrial ribosome. Reasoning behind the down regulation of this gene is not clear. The protein produced by the SPR28 gene functions in cell wall organization and biogenesis. Down regulation of a gene with this function was also not expected. Contrary to our hypothesis, these two genes do not produce proteins with similar function nor do they produce proteins functioning to aid oxidative stress. Genes found to be up regulated were CUE3 (locus tag YGL110C), FMP43 (locus tag YGR243W), HXT1 (locus tag YHR094C), TOF2 (locus tag YKR010C) and GTO3 (locus tag YMR251W). Genes CUE3, FMP43 and GTO3 have unknown functions according to Magic Tool gene function list. Upon further research CUE3 has been shown to possibly facilitate intramolecular monoubiquitination while GTO3 has been hypothesized to function as an omega class glutathione transferase. Glutathione transferases have been shown to be crucial enzymes in the cell detoxification process by catalyzing the detoxification of electrophilic substrates with glutathione (Dourado 2008). Therefore GTO3 gene up regulation could be a direct result of the yeast compensating for zms2Δ mutation. The GTO3 gene up regulation was the only gene that followed our hypothesis of the seven genes up or down regulated. Due to these findings our hypothesis was not supported by the data. There seemed to be little to no correlation in the up and down regulated genes and the zms2Δ mutation: the exception being the GTO3 gene.
The reason that there was no real correlation in the up and down regulated genes and the zms2Δ mutation could have been caused by errors that could have occurred during the experiment. The microarray slides were made by humans which leaves plenty of room for error in itself. In the experimental steps we may not have used enough cDNA to hybridize the microarray slide. The pipettes that we used to put the cDNA onto the slides may not have been calibrated correctly, which may have cause experimental error. The correct concentrations of each solutions for the hybridization and washing steps may not have been used. After the microarray was scanned the information needed to be manually girded, which could include human error, either not girding an entire gene or enclosing too much outside of the gene in the grids. The girding was done by four different members of our group who may have girded their section of the microarray differently causing the whole microarray to not be uniform. Finally, when analyzing the data problems arose such that the microarray slides were unable to be exactly standardized. The four microarrays observed did not have an equal mean at zero and were either above or below. This affects the determination of up and down regulated genes.
A more comprehensive study using additional microarray slides should be preformed to further analyze the microarray data to determine possible correlations between up and down regulated genes with oxidative stress functions. Statistical analyses, such as cluster analysis or T-test, on the data from this experiment could also be preformed for additional information. For example doing a cluster analysis to compare the zms1Δ and zms2Δ would show uniform up or down regulation conserved in both mutations.
Main Page Introduction Methods Results Literature Cited