INTRODUCTION

            DNA Microarray technology is an up and coming molecular biology technique which allows for the identification of gene or gene mutation sequences, the determination of gene expression levels when two populations of cells are compared, and the large scale examination of thousands of genes at the same time.  Since this method is relatively new, it is still experiencing procedural and technical difficulties. Despite the problems, with the increased usage and testing of microarray technology, it will not take long for a universal protocol, which will limit glitches, to develop.

A microarray is dependent on the creation of a gene library, and fragments of genes called expressed sequence tags (ESTs) based off an organism's genome .  These ESTs are spotted on a microarray slide by a robot, and acts like a target sequence for cDNA. cDNA for a microarray slide is made by reverse transcribing a population of RNA, particularly messenger RNA, from a sample organism and tagging the cDNA with a photosensitive dye.  The labeled cDNA is exposed to the microarray slide to which it hybridizes with the complimentary EST spots.  The slide is then scanned and the fluorescence of each spot is measured.  A strong fluorescence shows that the test cells’ cDNA fragments are bound to the given EST, and in consequence it is considered the active gene spot is expressed to a comparative degree in the organism.  On the other hand, if no fluorescence shows, then no cDNA hybridized to that spot and that gene is considered to be inactive in the cell (NCBI, 2004).   Figure 1 depicts the steps involved in the Microarray technique.

Figure1. Generalized Microarray procedure

 Naturally occurring oxygen is fatal to life forms. Although elemental oxygen is not harmful its derivatives such as as superoxide, hydrogen peroxide, and hydroxyl radicals may be lethal to organisms lacking the required mechanisms to process it (Micro Book).  Oxygen can damage lipids (mostly in plants), protein, and DNA causing mutations and even death.  In proteins activated oxygen can oxidize iron-sulfur centers therefore terminating enzymatic activity of the protein. In DNA oxygen radicals can cause nucleotide deletions,  mutations, and even extremely harmful double strand breaking . Despite these disadvantages oxygen's high oxidative potential has become especially useful in cells, and most specifically in the electron transport chain allowing aerobic organisms to create more energy than anaerobic organisms. Over time, evolution has provided methods for cells to properly process these toxic forms of oxygen in order to fully reap the energetic benefits of having oxygen within the cell.

This experiment focused on the microarray analysis of the ZMS1Δ gene in Saccharomyces cerevisiae. The gene ZMS1Δ has been identified as a gene that encodes a putative transcription factor (Lu et al., 2005).  Its product is involved in the transcriptional control of genes in cellular genetic compartments, respiration, and other functions.  In particular, ZMS1Δ regulates the ZWF1 gene, which allows for the production of glucose-6-phosphate dehydrogenase (Grabowski and Chelstowska, 2003).  This enzyme is important in assuring that there is adequate levels of  NADP+  in a cell. The coenzyme NADP+ prevents the cell from experiencing oxidative stress by eliminating detrimental activated oxygenic molecules such as the aforementioned superoxide, hydrogen peroxide, and hydroxyl radicals (McKersie, 1996).  To further understand ZMS1Δ’s involvement in controlling oxidative stress, a microarray experiment was set up which compared a wild-type strain of Saccaromyces cerevisiae, to a ZMS1Δ knock out strain.  It was hypothesized that the ZMS1Δ knock out strain would have altered levels of gene expression compared to the wild-type, and more specifically, genes encoding transcription factors implicated in the regulation of oxidative stress would show more differing gene expression.

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Josh Krueger       Kris Tillett       Gordo McGuire