INTRODUCTION

            Oxidative stress is caused by unwanted radicals in a cell that take away electrons from vital cell molecules and structures, such as DNA and proteins. To combat oxidative stress, cells use antioxidant enzymes such as catalase and superoxide dismutase to chemically alter the toxic particles into less harmful ones. In Saccharomyces cerevisiae, bakers yeast, some of these antioxidant enzymes are encoded by the genes zms1 and zms2. It was observed that when the zwf1 gene, which is known to be involved in reducing oxidative stress, in yeast was experimentally knocked out the zms1 and zms2 genes were up regulated in the mutants in order to compensate for this loss (Slekar, 2008). In this experiment we created three mutant knock outs; the zms1Δ mutant where the zms1 gene had been knocked out, the zms2Δ where the zms2 gene had been knocked out, and the zms1/2Δ where both the genes had been knocked out.

       This study analyzed three microarrays each containing one of the mutant knock outs and wild type yeast.  Genes in the zms1Δ,  zms2Δ, zms1/2Δ mutants were determined to be up regulated by comparing them to the wild type yeast's gene regulation. MagicTool was then used to quantify which genes were up regulated in the mutants when compared to the wildtype. The genes of particular interest were the ones that were up regulated in all three mutants. These genes were then identified using the NCBI database and their biological functions and processes were found. It is my hypothesis that the majority of the genes which are up regulated in all three mutant knock outs will be involved with some sort of process that helps to reduce oxidative stress in the yeast.

        This study examined all three knock out mutants zms1Δ,  zms2Δ, zms1/2Δ. It compared the regulation of the genes in each mutant in order to find the genes that were up regulated in all three mutants. This will hopefully provide studies in this subject with valuable information about how yeast compensates for the loss of the zms1 and/or zms2 genes.

METHODS

        The methods used for this study can be found in the following link (METHODS). The methods that differ from the link are the following. Four microarray slides were used, each containing one of the mutant knockouts, zms1Δ,  zms2Δ, zms1/2Δ and wild type.

RESULTS

        Any results not directly concerning the analysis this study is doing can be found in the following links (GROUP 3, GROUP 7, GROUP 8, and GROUP 9).

Figure 1: Box plots of zms1Δ,  zms2Δ, zms1/2Δ mutants after standardization. This accounted for any false readings of up or down regulation caused by the natural intensity of either the red or green dye which were used to label the wild type and mutant DNA on the microarray. The goal of this standardization was to get as close to a 1:1 ratio between all of the samples as possible. The left three are zms1Δ, the middle three are zms2Δ, and the right three are zms1/2Δ.

Figure 2: Shows the levels of regulation each gene had on each microarray. A value of 1.6 was required in order for the gene to be considered up regulated. This would mean that the gene had a three-fold up regulation according to the log2 scale that the values are in.

Table 1: Shows the biological process, location, and chromosome of all the genes that were found to be up regulated (see Fig. 2).

DISCUSSION

        This study was designed to identify the biological processes of the genes that were up regulated in zms1Δ,  zms2Δ, zms1/2Δ. Each gene had to be up regulated by at least three-fold, which would be equivalent to the value of 1.6 (Fig. 2). However, there were some discrepancies in the data that must be mentioned. Each gene that was used for the study had two values that were down regulated. The reason we chose to ignore these values is because the rest of the values for each gene were either very close to 1.6 or well above 1.6. As a result I strongly believe that these points were most likely abnormalities in the microarrays, probably caused by the lack of DNA at that certain spot in the microarray. Another discrepancy likely to cause some problems with the interpretation of the data is the standardization of each microarray (Fig. 1). In order to have the best data for interpretation our standardization results would have an ideal ratio of 1:1. However, this data did not achieve that ratio which may cause some genes to look more up regulated than they really were and some genes to look more down regulated than they really were. The data was close enough to make these errors less of a factor on the interpretation of how genes were regulated than they could have been.

        After determining which genes were up regulated, the biological processes of these genes were found using the NCBI gene database in the hope of discovering whether or not these genes were up regulated in order to compensate for the loss of the zms1 and/or zms2 genes. These two genes were shown to be involved in the reduction of oxidative stress in Saccharomyces cerevisiae in previous studies, so it was hypothesized that most of the genes that were up regulated in all of the mutants would have a biological process that influenced the reduction of oxidative stress.

        After researching the biological processes of each gene it was found that out of the six genes that were considered to be up regulated only two of those genes had biological processes that were possibly related with oxidative stress. The first gene was YDL120W, it was used by yeast to regulate mitochondrial iron accumulation and interacted with electron transport chain components that may influence respiration. The regulation of iron is especially interesting because metals, such as iron, as a direct result of their ability to either accept or donate a single electron, can catalyze reactions leading to the formation of a reactive radical or some other kind of reactive oxygen species. It also is thought to have some influence with mitochondrial respiration, specifically the electron transport chain, which is another pathway for the formation of toxic reactive oxygen species. The second gene was YNL234W, it was found to have a function similar to globins and a heme-binding domain. Heme's utilize iron molecules in order to transport diatomic gases, such as oxygen, detect diatomic gases, catalyze chemical reactions, and serve as a source or sink of electrons. Globins, globular proteins, are used for many different functions in cells, one of these is to transport of molecules in and out of the cell. Which indicated that the gene may be up regulated in order to transport the products of antioxidant enzymes out of the cell. Another possible explanation for its up regulation, is that globins are also used to catalyze metabolic reactions which can sometimes form reactive oxygen species. However all of these possible reasons for the gene being up regulated are only speculative in nature and cannot be confirmed until additional research is done into finding what type of globular protein this gene encodes for.

        Since only two up regulated genes that were possibly involved with biological process of reducing oxidative stress were found, more research must be done in order to support my hypothesis. In order to support my hypothesis a larger amount of genes and samples must be examined. Also, because two of the genes that were found to be up regulated had unknown biological processes research into their functions in yeast cells would also be useful to this study. The genes in question may actually be found to have some level of involvement with the reduction of oxidative stress, which would definitely provide support for my hypothesis.

LITERATURE CITED

Valko M, Morris H, Cronin MT (May 2005). "Metals, toxicity and oxidative stress". Curr. Med. Chem. 12 (10): 1161–208.

Seaver LC, Imlay JA (November 2004). "Are respiratory enzymes the primary sources of intracellular hydrogen peroxide?". J. Biol. Chem. 279 (47): 48742–50.

Slekar, KH., Presentation:  “A Genetic Study of Anti-Oxidant Factors in Yeast.” James Madison University, (2008).