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Discussion

  Microarray analysis was used to analyze the expression patterns of Saccharomyces cerevisiae.  We examined the expression patterns of ZWF1 knockout yeast strains that had two additional genes, ZMS1 and ZMS2, removed as well.  These genes had been previously implicated by Dr. Kimberly Slekar as playing a role in the oxidative stress response in yeast.  ZWF1 knockouts have a poor response to toxic oxygen molecules and are methionine auxotrophs.  ZMS1 and ZMS2 are suspected to be zinc finger transcription factors that interfere with the cells' antioxidant activity even further by interfering with response genes. Gene products from ZMS1 and ZMS2 suppress antioxidant genes, so we hypothesized that if ZMS1 and ZMS2 are not present yeast strains, the yeast will express a higher level of oxidative response genes.  An up regulation of sets of genes in double knockouts is expected and expression patterns of these genes were compared with ZWF1 knockouts with wild-type ZMS1 and ZMS2 genes.

  A total of 838 genes were analyzed present in grids 9 and 10 of two slides containing ZMS1 and ZMS2 knockout strains.  Wild-type and double knockout RNA that was used had a purity level, an A260nm/A280nm value, of 2.07 and 2.19, respectfully (Table 2). Both the wild-type and double knockout RNA were verified to have no RNA degradation by agarose gel electrophoresis.  The presence of the 28S and 18S bands for both RNA samples indicated this observation (Figure 1).  We identified seven genes that were up regulated in both of the two slides.  Standardization of the dye intensity ratios present in these two grids was performed to efficiently observe genes that were either up or down regulated.  Up regulated genes that were identified were YKL008C, YLR014C, YLR394W, YLR354C, YOL099C, YOR162C, and YGL212W (Table 4).  No genes were found to be down regulated in both of the slides that were analyzed.  Only one gene, YOL099C, was identified that did not have its biological function characterized yet.

  Two of the genes, YLR014C and YOR162C, have known transcription factor activity.  The gene YLR014C, the PPR1 gene, has been noted to have a role in starting transcription and the product of this gene, PPR1p, may induce the expression of the URA3 gene, which is a part of the uracil synthesis pathway (Patzoid 2001).  A gene such as this could stimulate a higher expression of URA3, which could play a role in oxidative stress.  The other transcription factor gene, YOR162C representing YRR1, is a drug resistance regulator gene that plays a role in driving the selenite stress response in yeast (Salin 2008).  This selenite stress response, the response seen when concentrations of selenite differ, has known to aid in interpreting the transcriptional networks in yeast for adaptation of a cell to its environment.  This gene, YRR1, may be helpful in mapping the complex networks on how its transcription factor ability has a role in oxidative stress. 

  Another gene found to be up regulated in double knockouts was YLR354C, which is the TAL1 gene.  The TAL1 gene is pentose phosphate shunt, which is considered a part of the pentose phosphate pathway.  This pathway converts glucose-6-phosphate into a sugar that is needed to go about cellular processes.  TAL1 is a basic helix-loop-helix protein that has been seen to regulate activation of endothelial cells through binding either directly or indirectly to DNA sequences in vital target genes (Tanaka 2008).  Up regulation of TAL1 could excite the pentose phosphate pathway in the absence of ZMS1 and ZMS2 to help in eliminating oxidative stress.  YKL008C, the LAC1 gene, is involved in the synthesis of ceramide by working in combination with another gene LAG1 to regulate the activity of ceramide synthase, the gene that produces ceramide (Kageyama-Yahara 2006).  This is important because in has an influence on the entire cell growth and death process due to the fact that ceramide is an important membrane component and is known to act in other ways including membrane traffic, apoptosis, growth arrest, and stress response.

  The last two genes, YLR394W and YGL212W representing CST9 and VAM7, have different functions that the other four genes.  CST9 is a replication protein that mediates homologus crossovers and it is involved in recombination of DNA during meiosis (Roeder 1995).  On the other hand, VAM7 is involved with protein transport in vacuoles (Yoh 1992).  These genes do not directly tie into oxidative stress but their function in mutants could be looked into to see if they play some role.  Otherwise, these two do not seem to have a large impact in the double knockout mutants.

  There is definitely room for future research on this topic.  Our results could have been skewed due to RNases degrading our experimental RNA or improper/ineffective labeling of the probes.  With this in mind, the experimental procedure could be altered to account for this or find new innovative methods for eliminating these factors.  We looked at only grids 9 and 10, so for more efficient data to be obtained we could replicate this experiment and look at more grids that would prove more beneficial toward identifying up or down regulated genes.  More genes could be identified performing this extended investigation as well as verifying the ones that we had already found being up regulated in double knockout mutants.  Lastly, newly identified genes could be placed into gene neighborhoods based on their chromosomal location which could give more evidence towards functionality.