Introduction
The pentose phosphate biological pathway, which consists of oxidative and non-oxidative phases, plays an integral role in cellular processes. A primary function of the pathway includes conversion of glucose-6-phosphate into simpler sugars, such as ribulose-5-phosphate, which act as carbon skeletons for necessary cellular compounds such as fatty acids and nucleosides (Kruger, 2003 and Slekar, 2008). Moreover, the initial conversion of glucose-6-phosphate to 6-phosphogluconate, performed by the enzyme glucose-6-phosphate dehydrogenase (G6PD), generates the majority of cellular NADPH. In addition to serving as a proton carrier in many enzymatic processes, NADPH acts as a cellular reducing agent, which plays an integral role in preventing oxidative stress (Juhnke et al., 1996).
Figure 1. The pentose phosphate pathway is responsible for conversion of glucose-6-phosphate into sugars, such as ribulose-5-phosphate, which are used to generate necessary cellular molecules.
Oxidative stress within the cell can occur as a result of one of several reactive oxygen species (ROS), such a hydroxyl radical (from hydrogen peroxide), which are formed during normal cellular metabolism (Juhnke et al., 1996). Without a reducing agent present in the cell, such as NADPH, free radicals can do major cellular damage to DNA, cell membranes, enzymes, proteins and have been linked to cancer, neurodegenerative and cardiovascular diseases (Slekar, 2008). Alternative sources of cellular reducing agents are therefore imperative to the viability of a cell with a mutation to the gene that encodes glucose-6-phosphate dehydrogenase, ZWF1.
S. cerevisiae, or baker’s yeast, is a single cell eukaryote which serves as an excellent model organism due to similarities within the biological processes of more complex organisms. Moreover, the entire yeast genome has been sequenced. Studies have indicated that yeast with a ZWF1 deletion (zwf1∆) exhibit sensitivity to hydrogen peroxide as well as methionine auxotrophy, indicating the yeasts inability to reduce the reactive species (Slekar, 2008). However, the stress response ALD6 gene has been shown to be an invaluable source of NADPH in cells with a ZWF1 deletion or mutation while strains with disruptions of both ZWF1 and ALD6 genes were co-lethal (Grabowska et al., 2003).
Two multi-copy suppressors of the zwf1∆ phenotype are the ZMS1 and ZMS2 genes. Although their function and method of suppression remains highly unknown, there is a possibility that ZMS1 and ZMS2 may be regulating trans factors associated with regulating ALD6 or other stress response genes. In order to further study the roles that these two genes play in the cell and the pentose phosphate pathway, microarray analysis of three mutant yeast strains will be performed. Mutant strain cDNA with a ZMS1 deletion, a ZMS1 and ZMS2 deletion (zms1/2) as well as cDNA from a wild type strain will be hybridized to a slide containing oligonucleotides from each gene in the yeast genome. Of all the data generated during microarray analysis, two grids, 7 and 8 containing a total of 836 genes will be analyzed using data from three hybridized microarray slides of the zms1 mutant and a single slide of the double mutant zms1/2. It is hypothesized that the there will be significant expression variance between the zms1 and zms1/2 mutants, with consistency between the zms1 triplicates.