Introduction        Methods        Results        Discussion        References        Individual Project - Karl

Results:

    In order to have the same levels of gene expression, due to cell conditions, fresh cultures were made the morning of the experiment for each strain of Saccharomyces cerevisiae.  This ensured that each strain were in the same stage of growth, so at any changes in gene expression would at least not be due to the cell's age.  Absorbance readings at 600nm were taken when the strains were initially cultured, and again just before the experiment began.  Table 1 shows the absorbance for each strain of yeast just before the RNA was isolated.

Table 1.  The absorbance of each strain of yeast at A₆₀₀.

    After RNA was isolated from the wild type and ΔZMS1 strains, the concentration and purity were determined using a nanodrop (Table 2).  The purity levels were determined by taking the absorbance level at 260nm divided by 280nm (A₂₆₀/A₂₈₀).  Purity levels of around 1.9-2.2 indicate a pure sample, our ΔZMS1 fell within that range, whereas our wild type RNA, with a purity value of 10.6, is considered impure.  Figure 1 shows RNA extracted from ΔZMS1 and wild type (lanes 4 and 5) respectively), in lane 2 there is a ladder showing bands for DNA, and then 28S, 16S, 5S, and 5.8S RNA.  Our samples appeared degraded, with our wild type possibly being contaminated with tRNA.

Table 2.  RNA purity and concentration determined using a nanodrop.

Figure 1.  RNA extracted from ΔZMS1 and wild type strains run on a 1.2% agarose gel.  Lane 1 contains a sample from group 1, lane 2 is a ladder, lane 3 is blank, lane 4 is our ΔZMS1 mutant, and lane 5 is our wild type sample.

    After determining that the wild type RNA we isolated was contaminated, we used RNA from another group.  The RNA for wild type and ΔZMS1 were reverse transcribed to produce cDNA to be hybridized on a microarray slide (#1360694) provided to us from GCAT, which were sent to Davidson College to be scanned.   Grids were manually aligned to each spot containing a gene using Scanalyze (Figure 2) and the expression levels were put into an excel spreadsheet.  Pixel intensities for the green (Cy3) and red (Cy5) dye were graphed in order to determine the normal distribution of data, in Figures 3-6.  If the slope equaled one then the dyes attached at the same rate, since none of the slopes were one, the data had to be standardized.  After subtracting out background pixel intensities and calculating the ratios of green dye to red dye the values were standardized and then analyzed in Magic Tool version 2.1. 

Figure 2.  Microarray slide #1360694 grid 3 hybridized with ΔZMS1 cDNA, labeled green with Cy3 dye, and wild type cDNA labeled red with Cy5 dye.

Figure 3.  Microarray slide #1360694 from group 2, with green  (Cy5) intensity on the x-axis and red  (Cy3) intensity on the y-axis.  The data is skewed towards green.

Figure 4.  Microarray slide #1360695 from group 1, with red (Cy3) intensity on the x-axis and green (Cy3) on the y-axis.  The data is skewed towards red.

Figure 5.  Microarray slide #1360722 from group 4, with red (Cy3) intensity on the x-axis and green (Cy5)  on the y-axis.  The data is skewed towards green.

Figure 6.  Microarray slide #1360727 from group 9, with red (Cy3) intensity on the x-axis and green (Cy5)  on the y-axis.  The data is skewed towards red.

Figure 7.  Standardized intensity ratios of the different slides after they were normalized.  From left to right the columns are of the following arrays: slide #1360694 top, slide #1360694 bottom, slide #1360695 top, slide #1360695 bottom, slide #1360722 which only did one, slide #1360727 top, and slide #1360727 bottom.

    There were 5 genes which we found to be distinctly up regulated within grids 3 and 4, and 5 genes which we found to be down regulated (Figure 8).  Table 3 shows the molecular function of the genes as well as cell location and reading start site and chromosome; two chromosomes: 4 and 13, have 3 genes each.  Figure 9 shows correlations between changes in expression levels in ΔZMS1 mutants compared with wild type.

Figure 8.  Gene expression ratios for ΔZMS1.  Negative numbers mean the gene is down regulated or express less compared to wild type, while positive numbers mean the gene is up regulated compared to wild type.  From left to right the columns are of the following arrays: slide #1360694 top, slide #1360694 bottom, slide #1360695 top, slide #1360695 bottom, slide #1360722 which only did one, slide #1360727 top, and slide #1360727 bottom.

Figure 9.  Circle diagram of gene expression correlations with a threshold of 0.5.  YAL017W, YDL118W, and YDR165W had similar patterns of down-regulation.  The genes YPL051W, YOR343C and  genes YML120C, YJL052C have similar patterns of up-regulation.

Table 3.  Gene information for the selected genes that were consistently up and down regulated.

Noble Egekwu - egekwuni@jmu.edu    Karl Gorzelnik - gorzelkv@jmu.edu    Jonathan Baugher - baughejl@jmu.edu