Oxygen is the third most abundant essential element in the universe. Oxygen (O2) is very reactive and capable of degrading organic molecules. O2 can easily generate very toxic byproducts such as O2-, H2O2, OH- that can destroy cellular constituents. Oxidative stress is caused by an overproduction of reactive oxygen species (ROS) which leads to biological machinery’s inability to readily detoxify the reactive intermediates or easily repair the resulting damage (2). ROS is beneficial to human body in limited amounts, as they are used by the immune system in fighting against pathogens. ROS is also used in cell signaling pathways (2). However, severe and uncontrolled cases of oxidative stress can lead to essential component damage such as DNA, RNA and protein degradation (2). Several enzymes are involved in maintaining the ROS balance in the human body by staying in a reduced state. Several human diseases such as Alzheimer’s and Parkinson’s disease are associated with oxidative stress (1).
Antioxidants are molecules capable of slowing or preventing the oxidation of other molecules, therefore, reducing the amount of reactive oxygen radicals present in the body. NADPH is a major source of electrons for the cell. These electrons are used by antioxidants to reduce molecules that have been oxidized by oxygen during oxidative stress. The pentose phosphate pathway involves the enzyme G6PD, which converts glucose 6-phophate into 6-phosphogluconate while reducing NADP+ into NADPH. When G6PD is not present in sufficient amounts, the body produces less NADPH; therefore, the organism becomes more vulnerable to oxidative stress consequences.
In order to better understand the G6PD, NADPH and oxidative stress pathway, Saccharomyces cerevisiae, commonly known as Baker’s yeast, was chosen due to several factors. S. cerevisiae is a single-celled eukaryote that contains antioxidant genes that are typically present in all eukaryotes. Also, S. cerevisiae is a facultative anaerobe that can be easily manipulated for the study and has a sequenced genome. S. cerevisiae contains a homolog G6PD gene known as ZWF1, which is responsible for regulating oxidative stress in yeasts. Therefore, during the absence of ZWF1 gene, the yeast exhibits signs of excessive oxidative stress. ZWF1Δ mutants are methionine auxotrophs and sensitive to oxygen. ZMS1 and ZMS2 are multi-copy suppressors of ZWF1 Δ. Past studies have shown signs of oxidative stress are repressed in yeast mutants with over expressed of ZMS1 and ZMS2 in the presence of ZWF1 knockout. However, single ZMS1Δ and ZMS2Δ knockouts do not show this ability suggesting a possible physiological role of ZMS1 and ZMS2 in oxidative stress protection.
The goal of the study is to compare expression levels between different yeast strains such as ZMS1Δ, ZMS2Δ, ZMS1Δ ZMS2Δ, ZMS1++ (over-expressed ZMS1), and ZMS2++. It is hypothesized that changes in gene expression levels will be observed between different yeast strains. In order to obtain the results, RNA will be isolated from yeast strains zms1Δ, zms2Δ, zms1Δ zms2Δ, ZMS1++, and ZMS2++ and WT. Microarray analysis will be used to study the gene patterns and help understand a possible role of ZMS1 and ZMS2 in suppression of ZWF1 mutation. Patterns of over-expressed and under-expressed genes involved in methionine pathway synthesis as well as other genes will be analyzed using cluster analysis and normalization techniques.
