Discussion
ZWF1 is an anti-oxidant factor found in yeast. ZMS1 and ZMS2 were found to be suppressors of the zwf1Δ. The goal of the experiment was to identify the effects of ZMS1 and ZMS2 in states of oxidative stress in S. cerevisiae. The purity of the isolated RNA was lower than the ideal pure values of 1.9-2.2. The sample could have been contaminated with different proteins or DNA. After running the RNA on an agarose gel, it had good quality with some DNA contamination. The 5.8S, 18S and 28S bands were distinct. No usable results from the zms2++ and wild type slide were found. Green dye was seen at the top of the slide but no red dye was found. The red dye could have oxidized before the slide was scanned. Specific binding was found at the top of the slide but nowhere else. Noise and random binding was found throughout the rest of the slide but yielded no usable data. Data from Fall 2004 was analyzed for other projects.
Common Promoter Regions Among Upregulated Genes:
The four upregulated genes that have similar transcription factors in their promoter regions have different functions. YPR158W is a putative protein of unknown function. YKL080W is a subunit of the V1 peripheral membrane domain, the proton pump. YAL015C is a DNA N-glycosylase that is involved in base excision repair. YPR168W is a subunit of the RNA polymerase II mediator complex. Future studies could research further into theses transcription factors to investigate if they have any involvement in oxidative stress and the production of anti-oxidant factors. Other research could be done to find more than four upregulated genes with a higher parameter than one standard deviation across numerous slides and compare their function to see if they are at all related.
Over and Under Expressed Genes in ZMS1∆ZMS2∆:
The most under and over expressed genes in the bottom portion of the106 ZMS1∆ZMS2∆ slide were determined using MagicTool (Table 4 and 5). These genes were then compared to the most over and under expressed genes in the top portion of the 106 ZMS1∆ZMS2∆. There were 21 commonly over expressed genes in the top and bottom of the slide. Those with the ten highest intensity values were used as the most over expressed genes between the two portions (Table 6). There were 11 genes found to be under expressed in both the top and bottom portion of the slide. The genes found commonly to be over and under expressed in both the top and bottom portion of the slide are more likely to biologically significant since the expression was duplicated. The expression levels of these genes between the top and bottom portion were not as similar as might be expected (Table 6 and 7). Since the top and bottom portion of the slide contains the same gene, one would expect the genes to have similar intensity values. The quality of the slides could explain the differences in expression levels between the same gene on the top and bottom portion of the slide. The somewhat poor quality of the microarry is likely due to experimental technique.
The genes which were under or over expressed in the ZMS1∆ZMS2∆ slide were then analyzed in the 104 ZMS1∆ slide to see how expression might change in these genes under different conditions. Overall, the genes that were over expressed in ZMS1∆ZMS2∆, were not over expressed in the ZMS1∆. These genes showed a decrease in expression under the ZMS1∆ condition (Figure 5). Of the genes that were under expressed in the ZMS1∆ZMS2∆ slide, only two also showed under expression in the ZMS1∆ slide as well. The genes showed to generally have an increased expression in the ZMS1∆ slide (Figure 6). The change in expression may be due to the presence of ZMS2.
The functions of the genes that were over expressed in the ZMS1∆ZMS2∆ mutant showed many ribosomal proteins (Table 10). It may be possible that the cell tries to compensate for the loss of transcription factors due to the knockout of both ZMS1 and ZMS2. In order to compensate, there may be something triggered to initiate transcription, which would explain the high levels of ribosomal proteins. Some over expressed genes were involved in energy production, which might also be related to the response of the double knockout. If the cell triggers additional transcription to occur, the cell would have to supply additional energy for this process. The genes that were under expressed showed some genes related in the process of cell division (Table 11). The reason for the under expression in these genes is unknown. Overall, the function of the genes that were over or under expressed in the ZMS1∆ZMS2∆ slide did not show any direct relation to oxidative stress. Further studies into the function of the commonly over and under expressed genes would be beneficial to the study at hand.
Other Protocols:
One of the biggest problems with a microarray project is getting the dyes to work properly. The two most common methods currently in use are the use of the capture sequence on the 5’ tail of the primers and the incorporation of the dyes into the cDNA by linking them to one of the dNTP’s, usually dUTP.
One way to limit problems associated with dye fading is to limit handling the dyes themselves. The protocol in the following link proposes that the dyes be mixed in with the RNA and primers during the protocol for making cDNA by reverse transcribing the mRNA. Using this method, there is only one hybridization method necessary, making the protocol shorter and potentially much easier to carry out.
Link: http://www.biotechnologycenter.org/hio/microarray/microprocedures/mapr.pdf
A problem that may be associated with microarray is a weak signal. The protocol below discusses the use of aminoallyl labeling of the cDNA. It is a method similar to using a dye linked to dUTP. Aminoallyl labeling of the cDNA involves using a dNTP that has a reactive primary amine that can react to form a bond with the dyes. However, the dyes are not incorporated until after the cDNA is made. This method would allow each strand of cDNA to have more dyes attached, and potentially produce a stronger signal.
Link: http://pga.tigr.org/PDF/BiotechniquesCookbook_II.pdf
Suggestions for Future Classes:
RNA degrades rapidly, and there are RNases every where. In the lab, it is stored for a week before it is reverse transcribed into the cDNA. To get good quality cDNA, it would probably be best to reverse transcribe the RNA right away, however, that would make the lab period too long. A good compromise for the class maybe to cancel the lab for 2 weeks during the normal lab time, but use that time on a Saturday or a Sunday to extract and reverse transcribe the RNA right away. Extracting and reverse transcribing the RNA to get cDNA takes up two lab periods, and in order to do these two procedures in one day, the class would have to start much earlier in the day.
ZMS1 has been researched and its role is known. The role of ZMS2 on the other hand is not fully known. Dr. Slekar thinks that it is a transcription factor because it codes for a zinc-finger protein. The best way to help her research may be to use the time spent on the microarray project to find out the role of ZMS2. Since there are a lot of problems with the red dye, it would be best to do single channel microarrays using only the green dye. A third of the class could do control slides or wild type yeast, a third could do ZMS2++ strain, and the remaining third could do an array on the ΔZMS2strain. It would be much easier to get data from this study since the green dye is what has shown up on the arrays in the past. While the class wouldn’t get as much data as a double channel microarray, the data from this project would probably be much more meaningful and much more easily processed and interpreted.