Discussion
In this experiment our goal was to use microarray analysis to check for variable gene expression in the ZMS2 mutant, a knockout mutant in the pentose phosphate pathway. The pentose phosphate pathway plays a major role in a oxidative stress pathway found in yeast. Three main approaches were taken with our data to see if any significant changes in the ZMS2 mutant could be found. Our data over all suffers due to a poor RNA (Figure 1) extraction and severe photobleaching (Figures 2 and 3). Our RNA was of such poor quality that no segment is visible on our gel. This is why the high cut-offs were set for both data sets to make sure the data was as real as possible.
The first approach was to observe all of the genes that were significantly repressed in both data sets. Among the significantly repressed genes in both data sets were genes YMR165C and YCL173C. These genes are involved in aerobic respiration and transport respectively and therefore did not pique our interests. This is because it does not seem to be involved in the pentose phosphate pathway.
The second approach was to observe the relative inductions in our mutant over the wild type and pick out some of the highest inductions we could find in both data sets. Looking at the green-green data displayed in Table 5, it can be seen that we had three genes that were highly induced in both data sets. One of these genes that shows high induction in the mutant is YLR383W (SMC6). It is over 256 fold induced in green-green data set and over 90 fold in the other! This gene deals with DNA repair mechanisms and with the structural maintenance of chromosomes. This may be significant because the ZMS2 gene is involved with the oxidative stress pathways in yeast. Since this is knocked out in our mutants perhaps this mutant compensates by inducing this SMC6 gene in order to recover from the increased DNA damage due to the decreased effectiveness of the yeast’s oxidative stress pathway. If free radicals are damaging DNA due to this loss-of-function mutant, then maybe this provides evidence that DNA repair has to step up to repair this damage. This provides that the ZMS2 mutants really are defective in their oxidative stress pathway due to this knocked out gene. The other two genes induced, YNL017C and YCR059C that also show significant expression on one microarray, code for proteins involved with vesicle mediat4ed transport and regulation of amino acid metabolism respectively which do not appear to be directly involved with this oxidative pathway mutation. The only gene highly induced in the red-green data set that is also significantly reduced in the green-green data set was gene YKL119C (Table 6). This gene is involved in protein complex assembly. Since it appears to not be clearly involved with the pentose phosphate pathway, no further information was gathered.
The final approach was to look at a DNA damage repair pathway and see if any of the proteins involved were significantly induced or repressed (Table 7). RAD17 is significantly repressed in the mutants and it is involved in the DNA checkpoint pathway.
Future Research:
Performing the microarray again with well extracted and plentiful RNA would make data analysis easier and more credible. Also, more pathways involved with DNA repair could be review with this new data to see if any significant changes in the mutant expression are present. It would also be prudent to check the activity of the reducing enzymes involved in dealing with free-radicals, such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase.