Home Introduction Materials and Methods Results Discussion Literature Cited
RNA microarrays were used to investigate the differences in gene expression between Δzms1 and wt yeast. Prior research has shown that over expression of ZMS1 can suppress ZMF1 mutations, which produce yeast with extra sensitivity to oxidative stress2. ZMS1 also may be a transcription factor because it has a zinc finger domain2. Hopefully this microarray analysis of Δzms1 yeast will highlight genes that may be regulated by ZMS1.
Yeast RNA expression can be affected by growth stages. Optical densities of the different yeast strains were obtained to determine if our yeast were all in the same growth stage prior to harvesting. The optical densities were all very similar and this indicated that the yeast strains were harvested during the mid-log growth phase (Table 1).
The RNA that was used to make the wt and Δzms1Δzms2 cDNA for the micro arrays was pure. Pure RNA has a ratio of A260nm/A280nm between 1.9 and 2.2. The purities of the total RNA from the wt and Δzms1Δzms2 yeast strains was in the upper limit of this range (Table 2). The total RNA was run on an agarose gel to assess possible degradation. Both the wt and Δzms1Δzms2 RNA had clear 28S and 18S bands, although the wt RNA had fainter bands (Figure 1). The presence of these clear bands suggests that the RNA was not degraded. Some contamination was likely present on the Δzms1Δzms2 RNA samples, as evident by the DNA smear.
The analysis of the microarrays may have been affected by dye bias. Dye bias occurs when one of the two dyes, used to label the cDNA, has stronger intensity than the other dye. Cy3 dyes are known to fade so microarrays that use C3 and Cy5 often have a Cy5 dye bias. Slides 13760695 and 1376094 had a red bias and had Δzms1 labeled with Cy3 (green) and wt labeled with Cy5 (red) (Figure 3). A dye swap was performed on slide # 13760722 by labeling the Δzms1 with Cy5 (red) and the wt with Cy3 (green). This slide, 13760722, had a green bias (Figure 3). Although there was not a systematic dye bias, with all the slides favoring one dye, each of our microarrays had some sort of dye bias, which can affect interpretation of the results.
The ZMS1 gene is a zinc-finger transcription factor that may be related to oxidative stress in yeast. Oxidative stress can result in free radicals in the cell. These free radicals can cause damage to the phospholipid bi-layer, proteins, and DNA2. Because ZMS1 is involved with suppressing ZMF1 mutations that cause oxidative stress phenotypes, genes that are differentially expressed in Δzms1 yeast may also be involved in oxidative stress. Analysis of the microarray data revealed several genes that may be differentially expressed in Δzms1 yeast (Table 4). We found 7 genes that were upreguated and 5 genes that were downregulated. Of these differentially expressed genes, four have no known biological function.
One gene of focus is YDL171C, or GLT1. It was found to be upregulated in Δzms1 yeast across all four slides. GLT1’s molecular function is glutamate synthase activity. Glutamate synthase produces NADH for the cell. In yeast, the glucose 6-phosphate dehydrogenase pathway uses NADP to form NADPH, which protects against oxidative stress by maintaining reduced glutathione in the cell. NADH is very similar to NADPH in that both are electron carriers. Maybe glutamate synthase NADH activity is also related to protecting the cell from oxidative stress by using the NADH in similar ways to the NADPH.
Further analysis of this microarray experiment should be performed. Collaboration with a biostatistician may be necessary to perform better normalization of the data and to adjust for the dye bias in the slides. A quartile normalization may be more effective than a mean normalization. Also, the Δzms2 and Δzms1Δzms2 microarrays need to be analyzed and correlated to the Δzms1 microarray. The controls should also be analyzed. For example, ZMS1 should be completely downregulated in the Δzms1 and Δzms1Δzms2 yeast while ZMS2 should be completely downregulated in Δzms2 and Δzms1Δzms2 yeast. The blanks on the microarray should also not reveal any differential expression if the arrays worked correctly. Criteria of a 3-fold increase in three of the four trials may also not be the best protocol for determining differentially expressed genes.
Microarrays are a powerful tool for finding differentially expressed genes, but ultimately other tests must be done to verify microarray results. Future experiments may use northern blots and real-time PCR to verify if these genes are truly differentially expressed in Δzms1 yeast. Also most microarray experiments use more than 4 microarray trials (most use 8) before they conclude that the genes are differentially expressed. Future microarray experiments may seek to minimize error by taking further precautions with RNases, and by optimizing hybridization temperatures.