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Introduction |
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Oxygen is a needed to sustain life for many organisms. However, oxygen can also be detrimental as well, especially in the form of reactive oxygen species (ROS). ROS are superoxide radicals, such as hydrogen peroxide, that are produced everyday as the products of normal metabolic processes like respiration. ROS serves many purposes such as immune defense, hormone synthesis, apoptosis, and fertilization (Donko et al 2005). Yet, ROS can also react with many macro molecules like DNA, proteins, and lipids and cause damage (Droge 2002). This damage is known as oxidative stress and involves tissue deterioration and dysregulation of signals. ROS also play roles in many diseases and ageing. Therefore, oxidative stress has become a medically significant problem Antioxidants are the means of protecting against oxidative stress and maintaining cell homeostasis. Antioxidants are able to remove ROS generated by metabolism. The source of these electrons is acquired from the molecule NADPH which is produced mainly through the pentose phosphate pathway. This pathway converts glucose into pentose phosphate sugars while manufacturing NADPH in the process through the reduction of glucose 6 phosphate. Glucose 6 phosphate is reduced by the enzyme glucose 6 phosphate dehydrogenase (G6PD). When there is an insufficient amount of G6PD, anemia and an increased susceptibility to oxidative stress occurs since little NADPH can be produced to act as a reducing agent (Bordin et al 2005).
To better understand the interaction between G6PD, NADPH, and oxidative stress, this pathway will be studied through the organism Saccharomyces cerevisiae, baker’s yeast. Yeast carries a homologous G6PD gene called ZWF1. When ZWF1 is deleted (zwf1Δ), the yeast will exhibit signs of severe oxidative stress. However, suppressors of this mutation called ZMS1 and ZMS2 have been identified through the work of Grabowska and Chelstowska and the Slekar laboratory. When these genes are over-expressed in zwf1Δ mutants, they are able to suppress the associated mutant phenotypes including oxidative sensitivity. Single zms1Δ or zms2Δ mutants do not show any sensitivity to oxdative conditions, yet a double zms1Δ zms2Δ does exhibit oxygen sensitivity. This suggests a possible physiological role for ZMS1 and ZMS2 in oxidative stress protection. Microarray analysis will be used to provide insight into the possible mechanism for ZMS1’s and ZMS2’s ability to suppress a zwf1Δ mutation. RNA will be isolated from yeast strains zms1Δ, zms2Δ, zms1Δ zms2Δ, ZMS1++ (over-expressed ZMS1 on a high copy plasmid), and ZMS2++ to compare expression level changes to a wild type strain. The changes in expression levels among all strains will be used in hopes of identifying a pattern that could lead into determining ZMS1’s and ZMS2’s potential role in oxidative stress protection. The changes in expression levels among all strains will be used in hopes of identifying a pattern that could lead into determining ZMS1’s and ZMS2’s potential role in oxidative stress protection by examining the most highly expressed and underexpressed genes along with the genes involved in the pentose phosphate pathway. In addition mean and median normalization techniques will be explored with this data in order to achieve better data in the future. |
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