Results:

Table 1. The optical density at 260 nm and 280 nm of the total RNA extracted from the wild type sample 2 and the ΔZMS1 sample 1 yeast cells.

Table 2. The concentration of total RNA extracted from the wild type sample 2 and the ΔZMS1 sample 1 yeast cells. Calculated using the O.D. 260 x ^{(40 µg/1000 µL)}/ _{1 O.D.} x 50. The purity calculated based on the ratio ^{O.D. 260}/_{O.D. 280}.

   Total RNA was extracted from the wild type-2 and ΔZMS1-1 yeast cells. The optical density at 260 nm for wild type-2 was determined to be 0.561and the optical density at 280 nm was 0.298. The optical density at 260 nm for ΔZMS1- 1 was determined to be 0.477 and the optical density at 280 nm was 0.242. Based on the optical density readings the purity was calculated (Table 1). The purity of wild type-2 was 1.88 and the purity for ΔZMS1- 1 was 1.97 . The concentration of each total RNA sample was calculated using the optical density reading at 260 nm (Table 2). Wild type-2 had a concentration of 1.122 µg/µL and ΔZMS1- 1 had a concentration of 0.954 µg/µL.

Figure 1. A 1.2 % agarose gel that was run at 100 volts containing the total RNA extracted yeast cells. Lanes 6 contains the wild type- 2 RNA and Lane 7 contains the ΔZMS1-1 RNA. The bands showing the DNA and rRNA, both 28S and 18S, of the samples are labeled.   

    Figure 1 shows the agarose gel that was run to determine if the RNA samples were denatured. The samples were run in Lanes 6 and 7 with wild type-2 in Lane 6 and ΔZMS1-1 in Lane 7. Lane 6 showed an abundance of DNA in its upper band while Lane 7 showed minimal amounts of DNA.  Both lanes showed two separate bands of rRNA which represent the 28S and 18S strands. The presence of the two separate bands and lack of smearing indicates the RNA was not degraded.

Figure 2. Image of 2004 microarray slide 104 in Scanalyze showing portions of bottom grids 5 and 6. ΔZMS1 is labled with red (Cy-5) and the wild-type is labeled green (Cy-3). Some impurities in the slide can be seen, note the bright fluorescent green spots caused by improper removal of the hybridization solution from the slide prior to scanning.

    After receiving the scanned data from Davidson on slide 13697727, it was determined that there was not enough data for analysis. Past data from the 2004 Bio 480 class was used for analysis instead. Microarray slide 104 was shown because it had the data from the same ZMS1 deletion (Figure 2). The yellow spots represent genes where there was equal expression between the wild-type and ΔZMS1 yeast. The red spots indicated over expression of the  genes in the ΔZMS1 yeast. The green spots indicated under expression of the genes in the ΔZMS1 yeast.

Figure 3. Pho85 related genes from 2004 microarray slide 104, with expression ratios transformed to log₂ which shows up/down regulation of the genes. YER059W (Pho85 cyclin V), YGL134W (Pho85 cyclin VII), YPL219W (Pho85p cyclin XIV), YGL215W (cyclin like protein that interacts with Pho85).

    Figure 3 shows four Pho85 related genes that were examined to compare the expression levels of the top and bottom grids of slide 104. In this figure the expression levels colored black represent those closest to the median of the data. The levels colored green show the genes with expression levels lower than the median while red shows those expression levels above the median. The median was found to be -0.96 for this portion of the data.

Figure 4. Plot of expression levels comparing the top and bottom of microarray slide 104 from 2004 for Pho85 related genes.  The expression levels were found using log₂ transformation. YER059W (Pho85 cyclin V), YGL134W (Pho85 cyclin VII), YPL219W (Pho85p cyclin XIV), YGL215W (cyclin like protein that interacts with Pho85).

    Figure 4 shows the same four genes illustrated in Figure 3. It compares the expression levels between the grids at the bottom and top of slide 104 for Pho85 related genes. This shows inconsistencies in the expression levels between the top and bottom grids.

Figure 5. The twelve most upregulated genes shown on microarray slide 104 from 2004 with ΔZMS1. The red line shown is a control gene on the slide, the twelve other genes are YBR100W, YDR274C, YEL065W, YHR216W, YIR040C, YJL099W, YJR128W, YML123C, YOR142W-A, YOR202W, YPR053C, and YPR122W. The threshold for the expression level was 2.5 for this figure.

Figure 6. The sixteen most downregulated genes shown on microarray slide 104 from 2004 with ΔZMS1. The genes shown are YBR086C, YBR113W, YBR254C, YDR215C, YDR437W, YEL039C, YEL041W, YFR032C, YFR053C, YGR112W, YJL132W, YNL019C, YNL132W, YOR009W, YPL141C, YOL104C. The threshold used for expression level for this figure was -7.3.

    Figure 5 and Figure 6 show the most up and down regulated genes on slide 104. Thresholds for levels of expression were used that would give less than 20 genes that had been up or down regulated. These figures, like Figure 4, show inconsistencies in expression levels between the top and bottom grids.

Extra Project

Project # 8

     Figure 5 and 6 show the 12 most up regulated and 16 down regulated genes respectively for slide 104 Fall 2004. The genes that are most up regulated were YBR100W, YDR274C, YEL065W, YHR216W, YIR040C, YJL099W, YJR128W, YML123C, YOR142W-A, YOR202W, YPR053C, and YPR122W. The known functions for some of these genes include ferrioxamine B permease (YEL065N), IMP dehydrogenase (YHR216W), involvement in chitin biosynthesis (YJL099W), inorganic phosphate transporter (YML123C), TyA gag protein (YOR142W-A), imidazoleglycerol- phosphate dehydratase (YOR202W), and putative homolog of human insulin-degrading endoprotease (YPR122W). From these functions, it is apparent that these genes are not related to the oxidative stress pathway being studied in this experiment.

    The genes that were down regulated the most on slide 104 from Fall 2004 were YBR086C, YBR113W, YBR254C, YDR215C, YDR437W, YEL039C, YEL041W, YFR032C, YFR053C, YGR112W, YJL132W, YNL019C, YNL132W, YOR009W, YPL141C, YOL104C. The known functions for some of these genes were probable transmembrane protein (YBR068C), probable membrane protein (YBR254C), iso-2-cytochrome (YEL039C), hexokinase I (YFR053C), mitochondrial protein with homology to the SURF-1 gene (YGR112W), and involvement in meiotic chromosome segregation (YOL104C). Yet again, from these functions it can be determined that these genes are not related to the oxidative stress pathway.

Table 3. Table showing the function and expression of genes from the ZMS2 slide from G5 2007. These eleven genes were the most under expressed genes found from the ZMS1 slide 104 2004 and the table examines whether they were under or over expressed on the ZMS2 slide.

Table 4.  Table showing the function and expression of genes from the ZMS2 slide from G5 2007. These four genes were the most over expressed genes found from the ZMS1 slide 104 2004 and the table examines whether they were under or over expressed on the ZMS2 slide.