RESULTS

RNA Isolation and Purification:

    The first step in determining the differences of genetic expression between the wild type yeast and mutant yeast involved separately isolating the total population of RNA out of the sample cells. Using yeast in the mid-log phase that had an optical density reading of 1.392 for wild type and 1.340 for mutant, it was determined that the concentration of yeast was approximately 3.5x10⁷ cells /mL (Table 1.). From this initial concentration, spheroplasts, which are fragile yeast cells that no longer have a cell wall, were made for both mutant and wild type. Breaking open the spheroplasts, and using a RNAsafe kit, RNA was isolated, DNA was captured and removed, and experiment ending RNases were degraded. Using spectrophotometry the concentrations of the final isolated RNA product were determined to be 0.294 ug/uL for wild type yeast, and 0.272 ug/uL for mutant yeast (Table 1.). on top of using the spectrophotometer to determine the thought to be concentration of RNA, absorbance readings at 260nm were divided by absorbance readings at 280nm to gather an initial understanding of the purity of the respective RNA samples( Table 1). Further analysis of the isolated RNA was then considered appropriate to gather a better understanding on what kind of shape the isolated RNA was in. Using the initial concentrations determined for both yeast samples, the same amount of RNA, approximately 3ug, was loaded separately into two15 ul agarose gel wells and electophoresed. From the gel run and picture taken, the 28s and 18s bands for ribosomal RNA were though to be distinct, no smearing was observed, and a bright patch near the beginning of the gel was noted (Figure 2).

Table 1. The determined optical density, concentration, and purity ratios obtained for both wild type and mutant RNA during isolation from the spheroplasts.

Figure 2. The agarose gel electrophoresis loaded with the mutant ZMS1 RNA in the bottom lane and wild type RNA in the second to bottom lane.

Microarray Synthesis and Data Analysis:

    After RNA isolation for the mutant and wild type yeast, cDNA was reverse transcribed with the incorporation of a dye capture primer. The cDNA made was then applied to a yeast Genome microarray chip along with a red dye that would attach to wild type cDNA and green dye that would attach to ZMS1Δ cDNA. The slides were then washed and sent off to Davidson college. After Davidson college returned the analyzed slides an image and pixel count of the microarray, which had its ESTs gridded and pixel count calculated using Scanalyze software, was generated (Figure 3). It was noted that on the microarray slide there was two mechanical scratches on one of the two genome sectors. From the pixel counts collected from the microarray, along with the pixel counts of a dye reversal group, two global normalization scatter plots were made. The first global normalization scatter plot was based off the green ZMS1Δ pixel count and the green wild type pixel count from the reverse group (Figure 4). This cross comparison was then fitted with a linear curve that had an correlation coefficient of .0069, and an angle to the x-axis that was almost zero. After the initial plot generated, it was decided that a global normalization plot should be done with the pixel counts for the green ZMS1Δ and red wild type microarray (Figure 5.). This global normalization had a correlation coefficient of .7106, but still had less then a forty-five degree angle relative to the x- axis (Figure 5.).

    After the global normalization of the green mutant/red wild type microarray, the empty spots of the gridded microarray were isolated and their average pixel counts were calculated along with the sample standard deviation (Table 2). From these obtained values the pixel counts two standard deviations away from the average was then calculated (Table 2). In order to consider the genes above the background noise, a pixel count in mutant genes had to be above 921 and the wild type genes had to have a pixel count above 615(table 2., figure 5.).

Figure 3. An actual image taken of the green ZMS1Δ and red wild type microarray with the apparent green tent and lack of red pigment noted.

Figure 4. From the microarray pixel counts a green wild type vs. green ZMS1Δ scatter plot was generated that has a line of best fit and correlation coefficient.

Figure 5. From the microarray pixel counts a red wild type vs. green ZMS1Δ scatter plot was generated that has a line of best fit and correlation coefficient. On this scatter plot the genes, empty spots, and  empty control spot background check cutoff was distinguished.

Table 2. the empty control spot background check average pixel count, standard deviation, and calculated cutoff point.

Induced and Repressed Genes of Significance:

    After the global normalization and empty spot back ground check, a ratio of green mutant to red wild type was calculated for the remaining genes not filtered out. The average ratio for these genes were around three,  but the thought to be determined ratio value, when taking into account the predominance of house keeping genes, should have been around one. By understanding the skew of the ratio, and determining the logarithms with the base two for the  genes in question, possible induced and repressed genes in the mutant ZMS1Δ were distinguished if they were thought to have a three fold level of induction or negative two fold repression (Table 3, Table 4). The genes that were thought to show a change in expression were then compared to gene ratios of the sister dye cross group and the green mutant/green wild type pixel count (Table 5). From this comparison, possible genes that showed a change in expression in multiple trials could be located (Table 5). From the Green ZMS1Δ  twenty-four genes were noted as having a higher level of expression in the mutant, while three genes were noted as being repressed( Table 3., Table 4.) From these initial genes thought to have an expression change, thirteen showed a pattern of induction across the three comparisons, while there were no similarities in repression (Table 5.)

Table 3. Genes considered as being induced in the green ZMS1Δ  microarray.

Table 4.   Genes considered as being induced in the red ZMS1Δ  microarray.

Table 5. Genes thought to show similar inducement across the three comparisons listed as the column name.

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Contact Information:

Josh Krueger       Kris Tillett       Gordo McGuire