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         The particular slide created by our group was broken and unable to be analyzed.  Therefore, we scanalyzed a slide from a group in a previous year who also worked for Dr. Daniel on the delta hexose.   

         To determine the growth phase in which the yeast cells would be harvested, the optical density of the mutant and wild type cultures were taken. From the OD readings seen in Table 1, it was determined that the yeast were in the mid log phase of growth. In this phase, there is approximately 1 x 10⁷ - 5 x 10⁷ cells per ml in the cultures. After the RNA was isolated from the cultures,  the absorbencies of the wild type and mutant cultures at 280nm and 260 nm were measured, Table 2. The purity of the RNA was then calculated. Using the measured absorbencies, the concentration of RNA in the solutions was calculated by multiplying the absorbance of the culture at 260 nm by 40ug/1000ul)/1 O.D. unit and by a dilution factor of 200. These results are shown in Table 3. To check the isolated RNA for degradation, 3uL of RNA from each sample was run on an agarose gel. The image is shown in Figure 1. The first lane was RNA isolated for the delta hexose mutant while the second was wild type RNA. In both lanes one band of RNA is clearly visible, which is most likely 28s ribosomal RNA. The RNA in both lanes looks partially degraded, but not so bad that it was unusable.

            After the RNA was hybridized to the microarray slide and scanned at Davidson’s College, the image of the microarray was received. A portion of the image is seen in Figure 2. Using the magic tool program, the slide was analyzed and expression ratios were computed. Displayed in figure 3 is a graph showing the genes that had the highest expression ratios, meaning that they were under expressed in the delta hexose mutants. These genes are described in Table 4. The next figure displays the genes with the lowest expression ratios. These genes are described in Table 5. Table 6 shows the expression ratios for 12 genes that were of interest to the project. These genes are further described in the discussion section. The final figure, Figure 6, is an image of a dendrogram taken from the hierarchal clustering preformed in magic tool.

RAW DATA

Table 1. Optical Density of yeast cell cultures.

Table 2. Absorbance readings of the RNA samples and the purity of the RNA (abs 260/abs 280)

Table 3. RNA concentration in yeast samples.

Figure 1. RNA from mutant and wild type yeast cells run on a 1.2% agarose gel. The arrow points to the our mRNA.  It should be noted there are several other bands present and therefore our RNA is not pure. Lane Contents: 1, blank; 2, Δhxt RNA; 3, wild type RNA; 4, blank.

MICROARRAY

Figure 2. Portion if the microarray picture analyzed. 

MAGIC TOOL

Exploration of Frequencies

Figure 3.  This plot shows those genes who expression level was above 9 for at least on of the two samples.  The values on the left reflect the expression level and it can be noted that many values are above 16, a four fold increase in expression.    The genes shown in red were sampled because they were similarly expressed in both the wt and mutants.

Figure 4.  This is a plot of the genes in discussed Table 4 and highlighted Figure 3. The 45 degree angle of the slope and zero value for the x-intercept shows that the genes sample were expressed the same in both the wildtype and delta hexose mutants. 

Table 4.  This table presents the information about the function and gene product of those genes sampled in Figure 3.

Figure 5. This plot shows those genes who expression level was below .0625, a four fold decrease in expression.  The values on the left reflect the expression level under this value in at least on of the two samples.  In comparison to Figure 3, the differences between Δhxt and wt are more clear.  The steep slopes show that several genes were differentially expressed at these low levels with more down regulation occurring in the wildtypes than the mutants.   The genes show in red were sampled because they were similarly expressed in both the wt and mutants.

Table 5.    This table presents the information about the function and gene product of those genes sampled in Figure 5.

Genes of Interest

Table 6. Average expression ratios for genes of interest. The average expression ratios between two slides and between each gene repeat was calculated, as well as the standard deviation for the data. The ratio represents red (mutant) over green (wild-type).

Hierarchical Clustering

Similarities in Gene Expression: Analyzed through Hierarchical Clustering

Figure 6. These three genes (listed below) were clustered hierarchically and analyzed through a computer program called MagicTool. The computer found and grouped the most related genes based on their gene expression. The gene names and descriptions are also listed below. The common characteristics to note are the fact that all the genes play a role in DNA repair or packaging.

YMR137C
ORF, Verified
Required for a post-incision step in the repair of DNA single and double-strand breaks that result from interstrand crosslinks produced by a variety of mono- and bi-functional psoralen derivatives; induced by UV-irradiation

YMR106C
ORF, Verified
Subunit of the telomeric Ku complex (Yku70p-Yku80p), involved in telomere length maintenance, structure and telomere position effect; relocates to sites of double-strand cleavage to promote nonhomologous end joining during DSB repair

YNL136W
ORF, Verified
Subunit of the NuA4 histone acetyltransferase complex, which acetylates the N-terminal tails of histones H4 and H2A