INTRODUCTION

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All aerobic organisms counteract reactive oxygen species with cellular antioxidants. Reactive oxygen species, such as hydrogen peroxide or superoxide anions, are free radicals that oxidize various intracellular macromolecules such as DNA, RNA, and proteins. The oxidation of these macromolecules results in the irreversible damage of cellular components and ultimate hemolysis of the cell. Oxidative stress plays a role in many pathological conditions such as cancer, Alzheimer’s, and Parkinson’s disease. (Immunity Today, LLC)

Antioxidants are molecules or compounds that counteract free radicals by donating electrons; antioxidants bind and reduce the oxidative agents, thus protecting cells from apoptosis caused by oxidative stress. Examples of antioxidants include intracellular enzymes such as glutathione peroxidase, superoxide dismutase or endogenous molecules including glutathione and thioredoxin. Essential nutrients, such as vitamins C and E, also function as antioxidants. Essentially, antioxidants react with free radicals in an attempt to prevent damage to cells within the body; they are kamikaze molecules willing to sacrifice their electrons for the overall health of the organism. (Immunity Today, LLC)

The Pentose Phosphate Pathway (PPP) plays a major role in the prevention of oxidative stress via the production of sugars and NADPH. In this pathway, Glucose-6-phosphate dehydrogenase is an enzyme encoded by the G6PD gene. This enzyme is active in nearly every cell and functions to facilitate carbohydrate processing. Specifically, the G6PD enzyme converts glucose to ribose-5-phosphate, a sugar essential in DNA and RNA synthesis. This process also reduces oxidized nicotinamide adenine dinucleotide phosphate (NADP⁺) molecules to NADPH. (NIH)

Saccharomyces cerevisiae, a facultative anaerobe, will be used in this experiment based upon its ideal characteristics. This model organism is an inexpensive and non-pathogenic unicellular eukaryote whose genome has been completely sequenced. This fungus is very easy to genetically manipulate and results are efficiently observed due to rapid growth and current knowledge of general pathways. Moreover, mutants are easily created via DNA transformation. Their stable state enables the study of mutations in haploid and diploid strains. S. cerevisiae is used in this experiment based upon its content of emblematic anti-oxidant genes present in all eukaryotes. (Botstein et al)

In yeast species Saccharomyces cerevisiae, the glucose-6-phosphate dehydrogenase enzyme is encoded by the G6PD homolog ZWF1. A mutation in the ZWF1 gene, noted as zwf1Δ, results in the inability of glucose-6-phosphate dehydrogenase to reduce NADP to NADPH. ZWF1 gene mutations contribute to the accumulation of oxidized NADP molecules increasing overall susceptibility for oxidative stress and subsequent pathology. The zwf1Δ mutant phenotype is characterized by aerobic methionine auxotrophy and increased sensitivity to oxygen and oxidizing agents. (Slekar)

Previous studies have indicated that a pho8 mutation suppresses a zwf1Δ mutant by potentially increasing the activity of ZMS1 and ZMS2, ultimately increasing the activity of stress response genes. ZMS1 and ZMS2 genes are multi-copy suppressors encoding potential zinc-finger transcription factors whereas PHO85 encodes a cyclin-dependent kinase. These suppressors play an obvious role in gene expression based upon previous growth studies. (Slekar)

If we, the scientific community, are to remedy devastating conditions created by oxidative stress in humans, we must first understand the role of suppressors in yeast. Discerning the role of suppressors on oxidative stress in S. cerevisiae will potentially facilitate similar comprehension of pathways in higher eukaryotes. The following experiment takes a forward step in understanding the role of suppressors in eukaryotes by observing gene expression during microarray analysis.

Specifically, this experiment considers gene expression in a Δzms1/Δzms2 double knockout strain. It is hypothesized that genes expressed in the Δzms1/Δzms2 will be up-regulated because of the absence of both Δzms1 and Δzms2. Typically, over expression of these genes results in suppression of the zwf1Δ phenotype.

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