A comparative Microarray Analysis of Up and Down Regulated

Genes from a zms2∆ mutant yeast strain versus a zms1∆

mutant yeast strain.

Abstract

Introduction

            The genes zms2 and zms1 found in baker’s yeast, Saccharomyces cerevisiae, are associated with oxidative stress relief (Slekar 2008). When over expressed in yeast they suppress the phenotypes of the zwf1 knockout mutant yeast strain (Slekar 2008). zwf1 codes for G6PD, which acts as a electron source for reactive oxygen molecules which cause oxidative stress. It has been hypothesized that the genes discovered to be up and down regulated in the previous study by Martin, Anderson, Plutt and Steidle will also be up and down regulated respectively. 

Methods

            The same experimental methods were used in this study as the previous study. The only difference in this study was that slides from group 1 top and bottom, group 2 top and bottom, and group 4 top were used, a total of 5 microarrays were analyzed. These slides were chosen because the microarrays compared the genome of the zms1∆ knockout mutant strain to the wild-type yeast strain. Only the genes found to be up or down regulated in the zms2∆ vs WT study were analyzed in this study. 

Results

Figure 1. A box-plot of slides from group 1, top and bottom, group 2, top and bottom, and group 4, top after standardization (named in order from left to right) after standardization using Magic Tool.

Table 1. Log2 intensities of the zms1∆ mutant of the up and down regulated genes found in the zms2∆ vs WT study. The highlighted shows the slides that determined the gene up or down regulated respectively.

             The microarrays were standardized, using Magic Tool, to make the means of all of the microarrays equal to one another and hopefully at zero on a box-plot. Figure 1, above, shows a box-plot of each microarray slide data after standardization. The mean of each microarray becomes closer to zero after standardization however each microarray’s data does not have a mean that is exactly zero, some are above or below. This allowed the genes to be analyzed. Table 1 shows the up and down regulated genes discovered in the zms2∆ vs WT study and the LOG² intensities of those genes found in the zms1∆ vs WT microarrays. If a gene is found outside of the box, in the box-plot (Figure 1) a gene is determined either up regulated (higher on the scale than the box) or down regulated (lower than the box on the scale). From the box-plot you can see that any gene equal to or above 1.3 are above all of the boxes for every microarray and can be determined up regulated for all of the microarrays. Also, any gene found equal to or below -1.2 can be determined down regulated because it is below every box for each microarray. None of the genes were found to be up or down regulated in all of the microarray slides. The highlighted portions of Table 1 are the slides that determined the genes up or down regulated respectively.

Discussion

               This study was done to compare the zms2∆ mutant to the zms1∆ mutant. This study was performed to help increase the knowledge of up and down regulated genes found in grids 13 and 14 of the zms2∆ mutant vs the WT study (Martin, Dustin, Anderson, and Steidle).

               It was determined that if a gene was up or down regulated in three out of the five microarrays slides it would be labeled up or down regulated respectively. This was determined because of all of the errors that can occur during experimentation of a microarray. Most of the problems come from human error, before the microarrays were scanned problems that could have taken place were not enough cDNA used to hybridize the slide or the correct concentrations of solutions used in each hybridization or washing step. After scanning, the microarrays were manually grided and genes could have been gridded too small or large. Also, the selected genes could have been tagged as invalid. Another problem that is apparent is the fact that the means of all of the slides are not equal to one another, therefore each gene found has a different intensity on each slide. This causes an uncertainty when determining a gene up or down regulated.   

              From Figure 1 you can see that both down regulated genes, YDR218C and YPL190C, fit this condition. These genes have functions that do not seem to be related to oxidative stress according to the zms2∆ vs WT study (Martin, Anderson, Plutt, and Steidle). The proposed hypothesis is correct for the down regulated genes in this study. The up regulated genes did not follow the hypothesis and only two of the five up regulated genes found in zms2∆ were up regulated in the zms1∆ mutant. YGL110C, YGR243W, and YHR094C were not up regulated. YGL110 has a function described in the previous study as facilitating intramolecular monoubiquitination, YGR243W has an unknown function, and YHR094C is involoved in glucose transporter activity (GCAT website, yeast gene info).  The functions of these genes do not seem to affect oxidative stress and therefore do not seem to be needed in the zms1∆ mutant. The two genes that were upregulated were YKR010C and YMR251W. YMR251W has an unknown function (GCAT website) and YKR010C is predicted to be an omega class glutathione transferase. These enzymes appear to be important in cell detoxification (Dourado 2008). Like the previous study this gene is predicted to be needed in an oxidative stress mutant yeast strain. This gene is needed in the zms2∆ mutant but it appears to be needed in the zms1∆ mutant.

            Future work in this field could be done on finding the function of the YMR251W gene and determining whether it’s function is related to oxidative stress or not. Also, comparing more microarrays could improve this analysis. Statistical analysis could be done on the data from both of the zms2∆ and zms1∆ studies could be done and compared. Also, analyzing the genes in grids 13 and 14 of the double zms1∆zms2∆ double knockout mutant vs WT would help determine if the genes found to be up and down regulated in the single knockouts concur with the results in the double knockout.  

Literature Cited

Dourado DF, Fernandes PA, Ramos MJ. Mammalian cytosolic glutathione transferases. Curr Protein Pept Sci. 2008 Aug 9 (4):325-37. 

Slekar, KH., Presentation:  “A Genetic Study of Anti-Oxidant Factors in Yeast.”