Abstract Introduction Methods Results Discussion References
After isolating RNA from the yeast cells, purity of the RNA was determined. However, the RNA purity was not optimal as seen in Table 1. This could be due to DNA contamination of the RNA samples. This result is confirmed by the gel shown in Figure 2. The bands labeled “DNA” in lanes 1 and 2 validate that there was contamination and affirm the decreased purity. The RNA bands are also very faintly present on Figure 2. This was expected because the concentrations of RNA were so low. There was slight degradation of RNA as well inferred by the weak smears on the gel.
The initial microarray of ZMS2 failed to exhibit correct binding of probes. (Data not presented) There were no distinct spots were seen along with random dye spots across the entire slide. This could have happened due to RNA degradation during isolation, failure of cDNA reverse transcriptase, drying out of the slide during hybridization, exposure of dyes to light, or physical damage to the slide during preparation.
Because the initial microarray did not work, our group analyzed slide 104 from 2004 which looked at ∆ZMS1 (Cy5) versus wild type (Cy3). As seen in Figure 3 this slide exhibited distinct spots where hybridization occurred. The light blue color indicates flagging of the spots using ScanAlyze software. This data was excluded from analysis. Flagging is needed because of large background noise that would give false expression levels. As observed in Figure 3, a large green signal is present throughout the lower portion of the slide. This signal is the result of errors in hybridization; the hybridization buffers could have dried during the hybridization process leaving behind the residue. Any signal in that region would be masked by the large background and would skew data analysis.
When looking at microarrays, it is important that both dyes have similar signal ratios for data to be interpreted correctly. Figure 4 shows the correlation between dye intensity of the two probes used in this microarray analysis. The equation y=0.5562 +4.5726 represents the correlation between experimental dye signal intensities. The correlation is not ideal (red line) and could be due to the red dyes (Cy5) fading with time. Initially the dyes were added in equal amounts but because the red fades there tends to be an abundance of green signal on the array. This can be mathematically normalized, however this study did not attempt to normalize the data prior to analysis. Instead of normalizing, critical values resulting in identification of induction or repression were adjusted to compensate for the faded dye. The data from previous year's slides was used to obtain more information in following areas: 1) To obtain RNA viability confirmation; 2) To determine induced and repressed genes in ZMS1 knockout mutant compared to WT yeast strain; 3) to determine the expression of genes involved in the methionine synthesis pathway for ZMS1Δ, ZMS2Δ and ZMS1 ZMS2 double-knockout.
RNA Viability Confirmation
In an effort to determine why the original microarray did not produce viable results, it was hypothesized that the RNA used may have become degraded. If degradation had occurred, little to no cDNA would have been produced to hybridize with the microarray. The results of the gel run to test for RNA degradation (figure 5) showed that some, but not all of the RNA was degraded. Some degradation can be assumed by the presence of faint bands. There is also some noticeable DNA contamination of the RNA sample. Based on these results, it seems unlikely that the microarray did not produce results due to RNA degradation.
Induced and Repressed Genes in ZMS1 Knockout Mutant Compared to Wildtype
Using MAGIC Tool, four genes were identified as being induced in ∆ZMS1 when compared to wild-type expression. (Table 2) Two of the genes (YFL026W and YNL145W) had functions related to pheromone alpha factors. Alpha factors are correlated to growth and division of cells and are play a role in G protein signaling. G proteins are involved in signaling pathways which could affect the expression of ZMS1 through alternative signaling pathways in the cell. YLR243W was interesting as well because it is a protein of unknown function but is thought to be a eukaryotic transcription factor. This could be related to ZMS1 in that previous studies have shown ZMS1 and ZMS2 to be potential zinc-finger transcription factors as well. If these oxidative stress transcription factors are mutated, the cell may in turn upregulate YLR243 to compensate during oxidative stress. From the expression profiles of the 4 genes (Figure 6) it could be inferred that the function of the unknown transcription factor (YLR243W) is similar to the alpha-factor receptor (YFL026W) or the pheromone a-factor (YLR243W) due to their similar expression patterns.
Using MAGIC Tool, ten genes were identified as being repressed by ∆ZMS1 when compared to wild-type expression (Table 3). The most interesting protein that was identified was YFL014W, which is a plasma membrane localized protein that is induced by heat shock as well as oxidative stress. This gene could be part of the oxidative stress response in which ZMS1 is involved. It was also found that the gene YLR327C was upregulated with almost exact expression patterns as the previously discussed heat shock induced gene (Figure 7). Because both are overexpressed in ∆ZMS1 experiments and have similar expression profiles, the two could have similar function. This could be another possible protein associated with oxidative stress response and ZMS1 in yeast.
Expression of genes involved in methionine synthesis
ZWF1 enzyme in the pentose phosphate pathway is responsible for the generation of NADPH molecules, which are the major source of electrons used by anti-oxidants to reduce reactive oxygen species (ROS). Absence of ZWF1 in yeast mutants results in increased symptoms of oxidative stress and yeasts’ inability to produce methionine resulting in methionine auxotrophy. This occurs because methionine synthesis requires significant amount of NADPH which is absent in ZWF1 knockout. Overexpression of ZMS1 and ZMS2 genes has been shown to suppress ZWF1 mutation; however, the mechanism is still unknown. It is believed that ZMS1 and ZMS2 may be involved in alternative NADPH synthesis pathway, which can help suppress methionine auxotrophy. It was predicted that genes regulating methionine synthesis would be repressed during ZMS1Δ and ZMS2Δ single mutants, however, methionine regulating genes should be induced during ZMS1 and ZMS2 double-knock-out. Therefore the genes involved in methionine synthesis are studied between different yeast strains (ZMS1Δ, ZMS2Δ and ZMS1Δ ZMS2Δ). Activation or deactivation of the alternative NADPH pathway may cause the methionine synthesis genes to be repressed or induced.
Results from the data analysis revealed three genes (SAM4, SAM2, MUP1) that were repressed in ZMS1Δ and two genes (SAM4, MUP2) that were repressed in ZMS2Δ (Table 4). Also, there was one gene (SAM2) that was induced in ZMS1, ZMS2 double mutant (Table 4). Past studies have shown SAM genes to be associated with Methionine Transsulfuration Pathway (Figure 8), which results in the production of Glutathione, a major thiol antioxidant in eukaryotic cells (3). The results from this experiment support the fact that during ZMS1 knockout SAM4 and SAM2 are repressed therefore, hindering the synthesis of glutathione, resulting in increased oxidative stress response. In double-knockout strain, ZMS1 and ZMS2 together are able to suppress ZWF1 mutation by activating alternative pathway for NADPH formation resulting in Methionine and glutathione production. Therefore, in the presence of both ZMS1 and ZMS2 mutations, the body is able to suppress oxidative stress symptoms and methionine auxotrophy. The results indicate some correlation between genes that are expressed and induced between different yeast strains. However, further research such as RT-PCR is needed to confirm these results.
Figure 8. Overview of the Methionine Transsulfuration Pathway (3)