Results

Wildtype and ZMS2++ mutant yeasts were obtained for RNA extraction.  The wildtype strain had an A600 of 0.727 while and A600 of the ZMS2++ strain was 1.05, indicating that both were in mid-log phase (Table 1).  A 1.2% agarose gel was used for gel electrophoresis to determine the quality of the RNA obtained (Figure 1).   The RNA for the wildtype yeast did not appear on the gel and the bands of the RNA from the ZMS2++ strain were very light.  The top of the lane that had been loaded with RNA from the ZMS2++ yeast strain showed some DNA contamination.

 Table 1.  The absorbance readings of liquid yeast cultures at 600nm on a spectrophotometer.

Figure 2.  A 1.2% agarose gel was used for gel electrophoresis to determine the quality of the extracted yeast RNA.  Lane 1 was loaded with RNA extracted from wildtype cultures and lane 2 was loaded with RNA from ZMS2++ cultures.  Arrows are pointing at the 28S, 18S, and 5.8S rRNA bands.

              The concentration and purity of the RNA used was calculated using a spectrophotometer at 600nm (Table 2).  The concentration of RNA from wildtype and ZMS2++ strains were 0.023 ug/uL and 0.027ug/gL, respectively.  These concentrations were too low, so RNA from another group was used with respective concentrations of 1.122 ug/uL and 0.338ug/uL. 

  Table 2.  The concentrations and purity of RNA obtained from wildtype and ZMS2++ yeast strains.  The concentration of RNA in our extractions were too low, so RNA from another group was used.

            The microarray that had been hybridized by our group was not useful for determining data.  The top of the slide had faint green spots showing where the cDNA from the control wildtype yeast had hybridized (Figure 2).  There were, however, very few red spots on this portion of the array.  Other areas of the array showed no specific hybridization by cDNA with either stains (Figure 3).  Most of the spots on these portions of the microarray slide appeared to be random binding.  

Figure 3.  The top grids of the microarray # 13697524.  There is some green signal with areas of smears of green dye.  The red dye is almost non-existent.  

Figure 4.  A middle portion of the microarray slide # 13697524.  More green dye than red is present, but both appear to be noise instead of useful signals.

Common Promotor Regions among Upregulated Genes:

After analyzing four microarray slides, YPR158W, YKL080W, YAL015C, and YPR168W were all found to be over expressed with a value greater than -1.09 throughout all four slides.  The genes were also found to have similar transcription factors in their promoter region as seen in Table 3.

 Table 3. Common transcription factors and their consensus sequence found on four microarray slides.

        The most over expressed genes in the bottom portion of the 106 slide ZMS1DZMS2D were found using MagicTool. The genes were narrowed down using those with only the greatest intensity values.  The 15 genes with values greater than 2.15 were determined to be the most over expressed in this portion of the slide (Table 4).  The under expressed genes in the bottom portion of the slide were found using MagicTool.  The intensity value for under expressed genes was less than -5.95. There were 28 genes that had identical expression values that matched the under expression criteria (Table 5). Since all of these genes had identical intensity values, the 15 most under expressed genes could not be determined.

        The expression of genes on the top and bottom portion of the ZMS1DZMS2D slide were compared to find the genes commonly over and under expressed. The Explore function of MagicTool was utilized to find genes which were over expressed in both the bottom and top portion.  An intensity value greater than 0.53 was used to find over expressed genes. A total of 21 genes was found and narrowed to 10 most over expressed genes (Table 6). The under expressed genes in both the top and bottom were found using an intensity value of less than -4.33.  A total of 11 genes were found to be under expressed in both the top and bottom of the ZMS1DZMS2D slide (Table 7).

The Expression of Genes in the ZMS1D Slide

        The genes found to be over and under expressed in the ZMS1DZMS2D slide were analyzed in another slide, the 104 ZMS1D. The expression of the genes in the ZMS1D slide were found using MagicTool and the expression file for the ZMS1D slide. The genes which were over expressed on the ZMS1DZMS2D slide were not over expressed genes on the ZMS1D slide (Table 8). As shown in Figure 4, the expression of the genes decreases in the ZMS1D slide. Two of the genes which were commonly under expressed in the ZMS1DZMS2D slide, were also under expressed in the ZMS1D slide (Table 9).  There were also two genes which were not found to have any expression value in the ZMS1D slide.  As shown in Figure 5, many of the genes under expressed in the ZMS1DZMS2D slide had increased expression in the ZMS1D slide. 

Function of Over and Under Expressed Genes

        The function of the genes which were over and under expressed were found using the GeneList from the 2004/2005 Yeast Chip Data.  Many of the genes that were over expressed were ribosomal proteins or were utilized in energy production (Table 10).  The genes that were under expressed did not show functions that are related.  Many of the functions of the under expressed gene were unknown (Table 11).

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