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Introduction

    Oxidative stress, the toxicity caused by active oxygen compounds, contributes to various debilitating disorders.  Active oxygen compounds interrupt cell function drastically without the presence of molecules that have the ability to reduce them.  This type of stress has been associated with Parkinson's, ALS, as well as many other neurological problems.  Oxidative stress has also been indicated to cause damage in heart cells and result in numerous heart problems.  Malfunctions in heart cells in diabetics have been seen to be caused by an excess amount of glucose which is an oxygen rich molecule.  However, researchers have discovered certain defense factors that aid in neutralizing active oxygen compounds (He 2008).  When in the presence of oxidative molecules, mouse heart cells were observed to increase production, or up-regulate, Nuclear factor erythroid-2 (Nrf2).  In a Nrf2 knockout mouse, a much higher presence of reactive oxygen species was observed.  The decrease in antioxidant activity in the absence of a Nrf2 gene resulted in increased damage to the heart.

Although studying oxidative stress has many implications, a smaller organism is more efficient for study of antioxidant genes and their regulation.  In this sense, S. Cerevisiae, baker's yeast, lacking the ZWF1 gene are a useful model.  Cells with the ZWF1 deletion are methionine auxotrophs and are incredibly sensitive to oxygen.  ZMF1 is thought to play a role in cellular defense against reactive oxygen species, so the knockout strain is more sensitive.   ZMS1 and ZMS2 are suppressor genes for ZMF1 that were first identified by Dr. Slekar.  When present in excess, or up-regulated, they act as suppressors for ZWF1 and contribute to the development of oxidative stress.  It is thought that they are zinc-finger transcription factors. If these genes are also knocked out in ZWF1 mutant strain, more antioxidant activity is observed.  However, more genes may be involved in this pathway, so it is necessary to observe changes between expression between strains.    For this study, all yeast used were ZWF1 knockouts lacking ZMS1 (ΔZMS1), ZMS2 (ΔZMS2), or both (ΔZMS1/ΔZMS2).  A strain only lacking ZMF1 was also used for wild-type comparisons.

            This study utilizes microarray analysis to determine the expression levels of genes in yeast strains. A microarray plate contains many short oligonucleotides that match a series of genes. The RNA from each yeast strain is harvested and reverse transcriptase is used to make cDNA. The newly created cDNA is labeled with a fluorescent probe and hybridized to the array.  The levels of fluorescence are measured and compared to observe changes in regulation between strains. In combination with statistical analysis, microarrays can show the expression of each gene in the yeast genome.

            In this particular study, gene regulation of ZWF1 knockout yeast strains will be compared.  A ZWF1 mutant with no other changes will be considered the wild-type.  Expression in the wild-type was compared to ZMS1 and ZMS2 knockouts using microarray analysis.  We hypothesize that the knockout strains will express a higher level of oxidative resistance genes in the absence of the suppressors for ZWF1.