ABSTRACT

Suppressors ΔZMS1, ΔZMS2, and ΔZMS1/ ΔZMS2 affect gene expression levels in Saccharomyces cerevisiae, as illustrated by ΔZMS1, ΔZMS2, and ΔZMS1/ ΔZMS2 knockout strains in previous studies. Typically, ΔZMS1/ ΔZMS2 suppress the phenotype exhibited by yeast suffering from oxidative stress. When these suppressors are no longer available to prevent oxidative stress, in knockout strains, variable genes become up or down regulated to compensate for the absence of the functional suppressor. For instance, ΔZMS1 may up-regulate genes to compensate for the loss of suppressor ΔZMS2, and vice versa. Gene expression also changes in a double knockout strain, where neither ΔZMS1 nor ΔZMS2 is present to provide relief from oxidative stress. This study was conducted to determine if there are commonly up or down regulated genes within three mutant yeast strains:  ΔZMS1, ΔZMS2, and ΔZMS1/ΔZMS2. At least one common gene was expected to be up or down regulated across all knockout strains. The PGS1 gene plays a role in phospholipid biosynthesis and was the only gene observed to be down regulated across all knockout yeast strains; no common gene was up regulated in all strains. The down regulation of PGS1 signifies that both the ΔZMS1 and ΔZMS2 suppressor play a similar role in the regulation of this gene. It is suspected that PGS1 plays a role in protecting yeast cells against the effects of oxidative stress based on its down regulation in mutant strains. However, further studies should be performed in order to secure the implications of these results.

METHODS

Data was used from slides created by the previous experiment using aforementioned Materials and Methods. This project is simply an extension of the previous project and data analysis is the only variable in that below results consider data from Saccharomyces cerevisiae ΔZMS1, ΔZMS2, and ΔZMS1/ ΔZMS2 knockout strains. Initial fluorescent values were acquired from excel spreadsheets of grids 11 and 27 on each individual slide 13760695, 13760728, and 13760728; the values were obtained using ScanAnalyze. Background intensities were subtracted from corresponding spot intensities to obtain values to be used in Magic Tool. Magic tool was used to create a  log₂2.50 expression scale of the data as well as to standardize the dye intensities. Statistical analysis was used to determine which of, at least, 25 genes were up- or down regulated. Genes that were up- or down regulated based on a log₂2.5 fold value were obtained and recorded for comparison. Finally, the specific gene names and corresponding biological functions were obtained from the GCAT website for further analysis.

RESULTS

TABLE 1. Up-regulated genes in three variable knockout Saccharomyces cerevisiae strains. An increase in expression level was determined using log₂2.5 and is represented below. No gene was up-regulated in all three strains. YIR038C was up-regulated in both ΔZMS1 and ΔZMS2 knockout strains only. Up-regulation in ORF genes YOR249C and YOR262W were seen in ΔZMS1 and ΔZMS1/ ΔZMS2 knockout strains. See Table 3 for appropriate gene names and their corresponding biological gene functions.

TABLE 2. Down regulated genes in three variable knockout Saccharomyces cerevisiae strains. Down regulated gene expression levels were determined using the negative value of log₂2.5 and are represented below. Only YCL004W was down-regulated in all three yeast strains. YBL071C and YNR063W were down regulated in both the ΔZMS1 and ΔZMS2 knockout strains. Only YER037W was seen to be down-regulated in ZMS1 and ΔZMS1/ ΔZMS2 knockout strains. See Table 3 for appropriate gene names and their corresponding biological gene functions.

TABLE 3. Gene names and corresponding biological gene function of up- or down regulated ORF

 Genes in ΔZMS1, ΔZMS2, and ΔZMS1/ ΔZMS2 knockout strains of Saccharomyces cerevisiae.

  TABLE 4. Up-regulated genes of variable Saccharomyces cerevisiae knockout strains. Data was obtained from grids 11 and 27 of slides 13760695, 13760728, and 13760729. An increase in expression level was determined using log₂2.5 and is represented below. No gene was consistently up-regulated in all samples. YIR038C was up-regulated in both ΔZMS1 and ΔZMS2 knockout strains only. Up-regulation of ORF genes YOR249C and YOR262W were seen in ΔZMS1 and ΔZMS1/ ΔZMS2 knockout strains. See Table 3 for appropriate gene names and their corresponding biological gene functions.

TABLE 5. Down-regulated genes of variable Saccharomyces cerevisiae knockout strains. Gene expression levels below negative log₂2.5 value are represented below. Data was obtained from grids 11 and 27 of slides 13760695, 13760728, and 13760729. Only YCL004W was consistently down-regulated in all yeast samples. YBL071C and YNR063W were down regulated in both the ΔZMS1 and ΔZMS2 knockout strains. Only YER037W was seen to be down-regulated in ZMS1 and ΔZMS1/ ΔZMS2 knockout strains. See Table 3 for appropriate gene names and their corresponding biological gene functions.

 Up-regulation of YIR038C was observed in both ΔZMS1 and ΔZMS2 knockout strains. Up-regulation in ORF genes YOR249C and YOR262W were seen in ΔZMS1 and ΔZMS1/ ΔZMS2 knockout strains. ΔZMS2 did not up-regulate the YOR249C or YOR262W gene. The YIR038C ORF gene was not up-regulated in the ΔZMS1/ ΔZMS2 knockout strain. The YIR038C ORF Gene, or GTT1, plays a role in glutathione metabolism while APC5, or YOR249C, plays a role in ubiquitin-dependent protein catabolism; the role of YOR262W, currently unidentified, is unknown (Table 3). (Table 1).

 A single gene was identified to be down regulated in all three strains of mutant yeast. ORF gene YCL004W, or PGSI, plays a role  in phospholipid biosynthesis. The ORF genes YBL071C and YNR063W, of unknown function, were down regulated in both the ΔZMS1 and ΔZMS2 knockout strains; they were not down regulated in the double knockout strain. The ORF gene of unknown function,YER037W or PHM8, was seen to be down-regulated in ZMS1 and ΔZMS1/ ΔZMS2 knockout strains. This gene was not down regulated by the ΔZMS2 knockout strain. Tables 2 and 3.

Gene up-regulation was not consistent throughout grids 11 and 27 on each slide; areas seen in black or green were down-regulated. (Table 4). In Table 5, only YCL004W as consistently down regulated in all grids throughout the three distinct slides. Black or plum colored areas represent genes that were slightly down or up-regulated (Table 5). 

DISCUSSION

Genes that were up-regulated in only ΔZMS1 knockouts suggests that the ΔZMS1 suppressor plays a role in gene expression. The GTT1 gene was up regulated in both individual knockout strains. This suggests that ΔZMS1 and ΔZMS2 function similarly to regulate the gene. The GTT1 gene, however, was not up-regulated in the double knockout strain. This indicates that, in the absence of ΔZMS1 and ΔZMS1, another suppressor may function to regulate the GTT1 gene responsible for glutathione metabolism. (Tables 1 and 3)

The APC5 gene plays a role in ubiquitin-dependent protein catabolism. The function of the currently un-named ORF gene, YOR262W, is unknown. Both APC5 and YOR262W were up-regulated in ΔZMS1 and ΔZMS1/ΔZMS2 knockout yeast strains. This indicates that the ΔZMS2 suppressor plays an insignificant role in the suppression of both genes. It may be that ΔZMS1 is primarily responsible for regulating both of these genes, or there may be another component dependent upon the ΔZMS1 suppressor to function; without ΔZMS1, the gene is no longer controlled. (Table 1 and 3)

The function and names of YBL071C and YNR063W have not yet been determined. Both ORF genes were down regulated in both individual knockout strains. This suggests that ΔZMS1 and ΔZMS2 play similar roles in regulating the expression of these genes. Surprisingly, these genes were not down regulated in the double knockout strain. This may indicate that another suppressor or molecule may regulate these genes only in the absence of both ΔZMS1 and ΔZMS1 suppressors. (Tables 2 and 3)

The function of the PHM1 gene is currently unknown. However, the gene is down regulated in ΔZMS1 and ΔZMS1/ΔZMS2 knockout yeast strains. This indicates that the ΔZMS2 suppressor does not play a role in the regulation of this gene. However, the ΔZMS1 suppressor is an essential part of the process responsible for the regulation of this gene. (Tables 2 and 3)

The gene of interest is the PGS1 gene that plays a role in phospholipid biosynthesis within Saccharomyces cerevisiae. This gene was down regulated in all three knockout strains. This trend suggests that ΔZMS1 and ΔZMS2 suppressors depend on one another to aid in the regulation of the PGS1 gene; no alternative pathway seems to be present to compensate for their absence. Basically, the PGS1 gene plays a vital role in the prevention of oxidative stress within different yeast strains.  (Tables 2 and 3)

Overall, it was very difficult to obtain genes that were consistently over or under expressed in the variable strains. The reason for this may be due to incomplete hybridization, RNA degradation, or numerous other mechanical or biological errors. In order to reinforce the ~5 fold change in expression levels, more grids should be analyzed. Further analysis should be performed on another set of knockout slides to determine if the results were victim to error or a true representation of ΔZMS1 and ΔZMS2 roles within the cell.

1)  Botsetin, D., Chervitz, SA., Cherry, JM. "Saccharomyces cerevisiae Information." Yeast (Budding, Fission, and Candida). 13 Sept. 2005. Saccharomyces Genome

                        Database. 19 Nov. 2008 <http://www.yeastgenome.org/vl-yeast.html>.

2)  Slekar, Kimberly. "A Genetic Study of Anti-Oxidant Factors in Yeast." James Madison University, Harrisonburg, VA. Oct. 2008.

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Theresa Russo