DNA microarrays are a fairly new but powerful tool in the field of biology. On the space of one standard microscope slide (called a "chip"), thousands of spots can be imprinted, each spot containing one gene for an organism. Using mutants of some organisms, yeast for example, where the entire genome has been completely sequenced, RNA can be isolated from the organism and hybridized to the slide. The RNA is reverse transcribed into cDNA, and then tagged with two fluorescent dyes: a green one and a red one. When hybridized to the slide, the cDNA's will bind to free nucleic acids on the slides. The slide is then read by a laser, and data is shown as amount of fluorescence for each dye. The ratio of fluorescence for each dye on each spot will tell the researcher how much each gene is being repressed or induced in the mutant vs. wild-type organism. Data analysis is extensive, but programs are available that simply the process of removing background fluorescence in order to obtain a more clear picture of what is going on.
The yeast Saccharomyces cerevisiae is a good organism to use for microarray analyses because the entire genome has been sequenced and many transcriptional products for what many genes encode are known. There are many databases available now that make it easy to find yeast genes and their known functions, along with BLAST searches that are helpful as well. Yeast are also good for these kinds of experiments because they are easy to culture in the lab and manipulate genetically, relative to more complex organisms. The yeast used in our experiment contain a mutation in the ZWF1 gene, a gene encoding glucose-6-phosphate dehydrogenase (G6PD) enzyme, and enzyme that is a critical first step in the pathway dealing with oxidative stress. The enzyme catalyzes the reduction of NADP to NADPH, allowing the cell to continue down the oxidative branch of the pentose phosphate pathway. Without this enzyme functioning, the cell has to adapt to oxidative stress, which causes damaging free-radical formation, in other ways in order to survive .The DNA microarray is a good way to analyze cell-wide changes in transcriptional activity due to this mutation, providing information about which genes are up-regulated or down-regulated as a result of this oxidative stress. From there, data analysis using programs such as Magic Tool can be used to group genes based on metabolic pathway relationships, providing information on how it is exactly that the cell copes with the stress of free-radicals when the G6PD enzyme is not functioning.
Free-radical formation is known to have many damaging effects to cellular processes. Highly reactive oxygen species create problems with DNA, possibly causing mutations that can result down the line in cell death or cancer. Cells have enzymes prepared to deal with this, superoxide dismutase for example, which forms hydrogen peroxide from these oxygen radicals (H2O2) that is then broken down into two molecules of water by the enzyme catalase (see picture below). As mentioned, left unchecked by these enzymes, free-radicals will negatively interact with DNA, causing strand breaks. With this in mind, we chose to analyze genes in our yeast that deal with DNA damage and repair, considering the mutants were less able to deal with oxidative stress and free-radical formation due to lack of G6PD. The induction or repression of several genes from the DNA damage and repair pathway were found and compared to our wild-type yeast data as well as another group's wild-type data. In induction and repression of several other genes were found and compared to other data sets, using a 4-fold cut-off (based on a log base 2 transformation) as a way to separate significant data from possible false-positives.
