Results

Discussion

Literature Cited

Introduction

Methods

Title Page

    Fresh yeast cultures were grown from a stock source for each of the mutant strains and wild type strain used in order to isolate RNA from cells which are at the same stage of growth. The yeast were allowed to grow to mid log phase by measuring the absorbance of the cell culture at an optical density of 600nm. Mid log phase for yeast is considered to be 0.4 - 1.7 absorbance at OD of 600nm and the optical density of the three strains used in this experiment are listed in Table 2 below.

Table 2. Absorbance readings for yeast cultures used to

isolate RNA samples for the reverse transcription into cDNA.

    Total RNA was isolated from the yeast cultures of all three strains and the purity and concentration of RNA samples used for the microarray slides analyzed in this experiment are listed in Table 3. The purity is a ratio of the Absorbance at 260nm to the Abosrbance at 280nm and was obtained using Nanodrop analysis. The concentration of the RNA is given in nanograms per microliter and was also obtained by Nanodrop analysis.

Table 3. Purity and Concentrations for the RNA samples of each group whose microarray data was analyzed.

    To be sure that the RNA samples isolated by each group were intact and not degraded, a 1.2% agarose gel was run and stained with Ethidium Bromide (Figure 2 and 3). RNA in Lane 2, 3 and 4 of Figure 2 and Lane 1, 3 and 4 of Figure 3 contain questionably intact RNA which is either a low concentration or degraded. There also appears to be DNA contaminant in all Lanes of Figure 2 and in Lane 2 of Figure 3.

                                                                    
Figure 2. RNA run on a 1.2% agarose gel. Lane 1 contains                                 Figure 3.  RNA run on a 1.2% agarose gel. Lane 1 contains
Δzms1 RNA isolated by Group 4 and Lane 2 contains Δzms2                               wild type RNA isolated by Group 3 and Lane 2 contains Δzms1Δzms2
RNA isolated by Group 4. Lane 3 contains Δzms2 RNA isolated                           RNA isolated by Group 3. Lane 3 contains Δzms2 RNA isolated
by Group 5 and Lane 4 contains Δzms1Δzms2 RNA isolated by                           by Group 2 and Lane 4 contains wild type RNA isolated by the same
the same group. Lane 5 and 6 contain RNA Δzms1 and wild type                         group.
RNA respectively, isolated by Group 1.

   After isolating the RNA, the selected RNA samples from Table 3 were reverse transcribed to produce cDNA which was then hybridized to microarray slides containing over 6500 yeast genes. All slides were set up to compare the mutant strain expression to wild type yeast expression. Figure 3 below is a sample portion of the microarray data which was then gridded in Scanalyze software (Blocks 7 and 8), and analyzed in Magic Tool version 2.1.

Figure 4. Portion of the microarray slide #1360722 hybridized with
Δzms1 cDNA labeled with Cy5 dye and Wild Type cDNA labeled
with Cy3 dye. Image obtained from Scanalyze software used for gridding.

    Data obtained from the scan at Davidson College were presented in an Excel file. Pixel intensities for both the Cy3 (CH1I) and Cy5 (CH2I) dyes for each gene were given, along with background intensities. Plots of the Cy3 vs. Cy5 intensities are expected to show a best fit line with a slope equal to one if the data are normally distributed. However, the plots in Figure 5 show that the data from group 4 (A) appears skewed in favor of Cy3 with the plot having a best fit line with slope greater than one (m = 5.0935). Similarly, the data from group 9 (D) is also skewed in favor of Cy3 (m = 1.6755). The data from groups 2 (B) and 1 (C) both show data skewed in favor of Cy5 with slopes less than one (m = 0.2378 and m = 0.01 respectively.

Figure 5. Graphs presenting the green and red intensities from the four slides analyzed before standardization. A. Group 4 Δzms1 labeled with Cy5 vs. wild type labeled with Cy3. B. Group 2 Δzms1 labeled with Cy3 vs. wild type labeled with Cy5. C. Group 1 Δzms1 labeled with Cy5 vs. wild type labeled with Cy3. D. Group 9 Δzms1Δzms2 labeled with Cy5 vs. wild type labeled with Cy3.
 

    In order to start analysis of the data, background intensities were subtracted off of the total intensities and any resulting negative or zero values were discarded. Then ratios of mutant to wild-type expression were obtained by dividing Cy5/Cy3 intensities. Data from each of the four microarray slides were then loaded into the software program Magic Tool for further analysis.   A log transformation of log to the base two was performed to have the data within a reasonable range for interpretation (from -16 to 16). Comparison between multiple slides is only reliable when the data sets have similar distributions so in order to visualize this, boxplots were created (Figure 6A). Since the distributions were not similar between the four microarray slides being analyzed, standardization to a mean of zero and a standard deviation of one of the data was required. After standardization the data were plotted again (Figure 6B) for confirmation that the distributions were approximately similar.

 A   B 
Figure 6. Box plots of the ratios for each microarray slide analyzed. A. Ratio values obtained after
taking the log base 2 of the ratios and before standardization. B. Ratio values obtained after standardizing
to a mean of zero and standard deviation of one. Image obtained from Magic Tool version 2.1 software.

    Genes were selected as significant if they showed consistent expression levels in all three Δzms1 trials and contrasting expression levels in the Δzms1/Δzms2 trial. Table 4 shows the twenty genes selected as significant along with the specific intensity ratios observed on each microarray slide. Table 5 shows those selected genes’ molecular function and the biological processes in which they are involved.      

Table 4. Genes with consistent expression patterns within three trials of ZMS1 mutants which have expression patterns in contrast to the ZMS1 and ZMS2 double mutant. The ORF name, gene name if known and chromosome number are given. Expression patterns are displayed in intensity ratios of mutant to wild type, where a negative ratio represents down-regulation and a positive ratio represents up-regulation.

Table 5. Biological processes and molecular functions of genes found to be differentially expressed between the ZMS1 mutant and the ZMS1 and ZMS2 double mutant, corresponding to the genes in Table 4.