Results

Yeast cultures initially obtained for experimentation included ZMS1, ZMS2, Wildtype (WT) 1 and WT 2. The amount of yeast culture acquired, the amount of SD Media used in the culture, and the absorbance of the yeast at 650nm was calculated for each of the four different variants of the yeast used. For WT 1, the actual grams of yeast culture acquired was 37.6g, the amount of SD media used was 60.4 g, and the absorbance of the yeast culture on the spectrometer at 650nm was found at 0.798. For WT 2 the respective values obtained were, 33.6g, 66.4g and 0.894nm. The ZMS1 value for the actual amount of yeast culture acquired was 42.7g, the amount of SD media was 57.2g, and the absorbance on the spectrometer at 650nm was 0.702nm. The respective values for ZMS2 were found as 40.0g, 60.3g, and 0.756nm. The obtained results are displayed below in Table 1.

Table 1. Table representing the Saccharomyces cerevisiae cultures obtained to perform experimentation. Table displays the amount of yeast culture used (g), the amount of SD media used in the culture (g), and the absorbance of the yeast culture, as measured by spectrometer, measured at 650nm. Four types of yeast variants were tested, ZMS1, ZMS2, Wildtype (WT) 1, and WT2.

The RNA to protein ratio of ZMS2 and wildtype 2 was measured via nanodrop. The nanodrop measurement, measuring RNA concentration in the yeast culture, was obtained at ng/uL and the ratio of the nanodrop reading absorbances obtained at 260/280, the purity ratio, was also recorded for both yeast cultures measured. The RNA concentration for ZMS2 was found at 381.1ng/uL and the absorbance ratio at 260/280 was found at 2.11. The RNA concentration of the Wildtype 2 variant of yeast was found at 408.3 ng/uL, and the purity ratio was found at 2.11. The obtained results are displayed below in Table 2.

Table 2. Table representing the RNA to protein ratio of ZMS2 and wildtype 2 measured via nanodrop. The concentration of wildtype 2 yeast was found at 408.1 ng/uL and the concentration of the ZMS2 yeast was found at 381.1ng/uL. The purity ratio at 260/280 of the wildtype 2 yeast was found at 2.11 and the purity ratio of the ZMS2 yeast at 260/280 was also found at 2.11

An RNA gel eletrophoresis was then run and obtained. The ZMS2 and the Wildtype 2 yeast data currently being examined were run in lanes 4 and 5 respectively. The gel was run at 18V for 25 minutes. The gel was then taken and washed according to methods protocol. The data was run with other similar data which can be later used to further examine results. The band at 28s and 18s were located and examined for further analysis. The gel is shown below in Figure 1.

Figure 1. RNA gel electrophoresis of Saccharomyces cerevisiae cultures of variants ZMS2 and Wildtype 2. Lane four displays ZMS2 yeast variant and lane five displays Wildtype 2 yeast variant. Gels were run at 18mV for 25 minutes. The gel was then washed as written in methods protocol. The gel is numbered left to right as lanes 1-8.

A microarray slide of the obtained data was then created. Due to reasons explained in discussion, data from Fall 2006 was analyzed for the remainder of the experimentation. An image of the data is displayed below as Figure 2. ZMS2 yeast was marked using red dye and Wildtype 2 yeast was marked using green dye. Areas where expression is present by both yeast variants is found as yellow. Slide displays 2 sets of the same data run.

Figure 2. Representation of microarray of ZMS2 and Wildtype 2 yeast RNA. ZMS2 yeast was marked with red dye and is displayed as red dots, wildtype 2 yeast was marked with green dye and is displayed as green dots. Areas with expression by both variants is displayed as yellow dots.   This is slide 726 from Fall 2008 ZMS2 labeled with red dye.

The microarray data was uploaded into Magictool analyzing software. The data was then organized into quantifiable results. A graph was created depicting the obtained results. The graphical data is shown below as Figure 3.

(a)

(b)

Figure 3. Graphical display of obtained results from Microarray of ZMS2 and Wildtype 2 yeast variants. (a) Represents the data as a function of baselog 2. (b) Represents shows the normalization of the data as a function of baselog 2.

Genes characterized as potentially important differences between the two variants were flagged and investigated. Several genes that had varied expression in the mutants were noted.  Upon further investigation, these genes were found to be linked to glycogensis. Differences in gene expression were noted in genes pertaining to glycogenesis to investigate if a pattern existed. This gene data was then then extracted and further analyzed. The genes were selected based on function rather than levels of expression.  The gene names, function, and level of expression in both ZMS2 and ZMS1 yeast variants compared to Wildtype yeast are displayed below in Table 3. ``

Table 3. Table depicting gene name, function, and level of expression of mRNA in  ZMS1 and ZMS2 knockout yeast compared to Wildtype yeast. Eighteen genes of interest were analyzed by comparing expression levels from the ZMS1 and ZMS2 knockout yeast to expression levels from Wildtype yeast. The gene expression levels were found by microarray analysis, using magic tool version 2.1, for ZMS1 and ZMS2 knockout yeast and Wildtype yeast.  Two slides were used per mutant, to account for dye bias.

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Discussion

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