DISCUSSION

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    Agarose gel electrophoresis results, of the isolated yeast RNA, showed successful extraction for ∆ZMS1/∆ZMS2 strain (Figure 1). The double knockout strain exhibited an acceptable purity value of 2.13. This value indicated that there were insignificant levels of protein in the sample. Contrarily, the wild type yeast strain showed partial RNA degradation. Sample contamination with RNAses may have caused degradation of the wild type strain.

    Microarray data received from Davidson College was subjected to ScanAnalyze. Initial mean normalization of the data was performed in order to compensate for non-biological noise. The average background intensity for the wild type strain versus the mutant strain was 981:219 for slide 13760727 (group 9) and 225:364 for slide 13760723 (group 5). Specifically, the mean normalization allowed for a more accurate comparison between genes within selected grids of the two different microarrays. Figure 2 shows that the adjusted mean values are all approximately zero using a log₂ scale. The values exclude any genes that were flagged while using the Scanalyze program.

    Initially, we were interested in 21 genes that were up-regulated. We then examined the consistency in up-regulation across all 4 grids; all genes lacking up regulation in at least 3 of the 4 grids were eliminated. From this we determined that 10 genes were up regulated (Table 1). The biological function for 3 of the 10 genes is currently unknown while the molecular function is known for only 4 of the 10 genes: MIG1, RRN7, YEF3, TRF4.

    MIG 1 encodes for a zinc finger protein that aids in the repression of glucose in yeast (Ostling et al, 1996). We found that this gene was over expressed in 3 of 4 grids. In regards to Dr. Slekars lab, the repression of glucose in the cell could have major implications in glycolysis and the citric acid cycle. However, the protein is mainly found in either the nucleus or cytoplasm, so it is improbable that Mig1p is involved in reducing oxidative stress in the mitochondria. It would be interesting to see if the Mig1 protein has any involvement in the cell with Glucose 6-Phosphate Dehydrogenase or any other enzyme in glycolysis.

    RRN7 was another gene that was highly up regulated in 3 of 4 grids. In the cell it functions as a transcription factor for RNA polymerase I activity. The up regulation of this gene would result in increased synthesis of RNA polymerase I, thus leading to the synthesis of the 28S, 18S, and 5.8S rRNA subunits. The over expression of RRN7 appears to have no direct function in the Pentose Phosphate Pathway.

    TRF4 also appears to be uninvolved in oxidative stress reduction, as its main function in the cell is directed towards mitotic chromosome condensation, according to the GCAT database. The Comprehensive Yeast Genome Database indicates that TRF4 is highly involved with poly(A) polymerase activity of pre-mRNA. From our results we saw that this gene was the most up regulated of all the genes examined. The high expression levels of this gene may compensate for the large amounts of RNA that need adenylation in the cell in order to be translated into protein.

    Another gene with known molecular function that was up regulated was YEF3. Although it was consistently over expressed, there was only 1 grid that demonstrated high expression of the YEF3 gene (Table 1). We were therefore unable to confidently identify YEF3 as a highly up regulated gene.

    Of the four genes that were determined to be down regulated only Met6 and MRPL19 had known biological functions. Met6 and MRPL19 are involved in methionine metabolism and protein biosynthesis, respectively.  The ∆ZMS1/∆ZMS2 yeast are methionine auxotrophs in that they must obtain methionine from the environment as they are unable to produce it themselves. In considering the auxotrophic nature of the yeast, it was interesting that the Met6 gene was down regulated.

    Moreover, oxidative stress damages many proteins that are necessary for specific functions throughout the cell. Without these proteins cells are unable to function properly and may die via hemolysis. MRPL19 plays a role in the synthesis of proteins that were previously damaged by reactive oxygen species.

    Some experimental errors occurred during probe hybridization. The microarray that was hybridized (slide 13760727) appeared to have a much more intense green dye compared to the red dye. Also, the green dye appeared to be dried up on the surface of most of the slide. This may have resulted from the salt drying on the slide or if the slide was not kept moist enough during the hybridization.

    Further, there were differences in gene expression within each of the 4 grids that were compared across the two slides. Variable genes may have been expressed due to a variety of problems including inconsistencies in the concentration of RNA used between slides, an error in the washing steps, or inadequate probe construction. Both the normalization and standardization of the data was an attempt to compensate for these differences. In the future, greater care should be used in probe construction to eliminate the different expression levels. Also, the manner in which the experiment is carried out should be done so more carefully to reduce the possibility of sample contamination with RNAses.

    Finally, further exploration of the Mig1 protein should be carried out to determine if its function has any impact on the repression of glucose in cellular respiration processes.

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