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After RNA was isolated from yeast cells, an agarose gel was performed in order to verify that good quality RNA was present. In the gel, clear bands were observed for 28s, 18s, 5s and 5.8s RNA, indicating that RNA isolation was successful (Figure 1). DNA band was also present in the isolated RNA sample. 2 left lanes are from another experiment ran on the same gel.
Figure 1. Agarose gel of RNA isolated from yeast. The isolates from this experiment are in the two right lanes. The first of the right lanes contains ZMS1++ RNA, the second ZMS2++ RNA. First band is DNA that was extracted along with the RNA. 2nd band is 28S rRNA, 3rd band 18S rRNA, last band 5S and 5.8S rRNA. The RNA concentration and purity for the yeast samples were determined using a spectrophotometer at 260nm and both 260nm and 280nm for purity. Figure 2 displays results.
Figure 2. RNA data of ZMS1++ and ZMS2++ used in the gel. ZMS1++ and WT samples were used for our microarray. The yeast samples were then hybridized onto Slide #129 and sent for processing. Since Side #129 had no useful data, Slide #104 was analyzed. An illustration of Silde 104 after processing is seen in Illustration 1.
Illustration 1. Grids 3,4,7,8 of Slide 104 fall 2004 showing useable results for microarray analysis. Spots amplified using Scanalyze (gain set at ~ 5). Slide 104 was used for microarray analysis due to inadequate data from Slide 129 fall 2007. Clustering Analysis Results The following results are from project investigating 15 highest and lowest expressed genes from Slide 104 and Slide 106 (unrelated to above gel). Table 1. Data from top half of slide 104 of fall 2004 with ZMS1 knockout mutant. Ratios were log transformed (base 2) in Microsoft Excel and grouped together with Magic Tool. The most overexpressed genes ranked from highest (top) to lowest (bottom). Underexpressed column ranked from highest underexpression (top) to lowest underexpression (bottom).
Table 2. Data from bottom half of slide 104 of fall 2004 with ZMS1 knockout mutant. Expression ratios were log transformed (base 2) in Microsoft Excel and analyzed with Magic Tool. The genes are ranked similar to above table 2.
Table 3. Raw data for above analysis was gathered from slide 106 Fall 2004, which is ZMS1 and ZMS2 double knockout. Log transformation was performed, Magic Tool was used to find genes with overexpression and underexpression. Data is ranked similarly to above tables.
While none of the same genes were found in any of the underexpressed or overexpressed catagories, a few common themes to the above tables are metabolism, meiosis and conjugation, and amino acid synthesis. Fructose metabolism is underexpressed on slide 104 (Table 1), in addition to glycerol and carbohydrate metabolism on slide 106 (Table 3). Sulfur metabolism, however, is upregulated on slide 106. Serine and histadine amino acid synthesis are downregulated on slide 104 (Table 1), likewise tryptophan synthesis is underexpressed on slide 106 (Table 3). Meiosis, conjugation, and growth genes were common elements in all the above tables. Pseudohyphal growth is downregulated in Table 1, as well as meiotic recombination in Table 2. Also in Table 2 a conjugation pheromone response gene is upregulated. Finally in Table 3 genes with functions in conjugation, meiosis, and chromatin modeling are underexpressed. Normalization Results Red and green intensities for the first 500 genes for all four microarrays were grouped together and their frequencies were calculated on separate histogram graphs. Figure 3 and 4 displays frequencies of green and red intensity values, respectively, without log base 2 conversion but background has been subtracted from these values.
Figure 3. Frequency of green intensities for all four microarrays. X-axis is green intensity value for all genes (CH1I-CH1B). According to histogram, the data is right skewed.
Figure 4. Frequency of red intensities for all four microarrays. X-axis is red intensity value of all genes (CH2I-CH2B). According to the histogram, the data is right skewed. Red and green intensity values for first 500 genes were then converted into their log base 2 values. Red and green values were then separately graphed into histograms to display frequency of each intensity. Mean and median normalization were then applied to the log base 2 values of both red and green intensities. Figures 5-8 graphically displays the log base 2 results of green and red intensities before and after mean and median normalization. For CH1 (Green) Intensities:
For CH2 (Red) Intensities:
Mutant to wildtype gene expression were then normalized using mean and median normalization. The first 500 genes of Monday and Tuesday's Slide #104 Top and Bottom microarrays were used. Mutant to wildtype gene expression was calculated for each gene, and mean and median normalization was then performed for each microarray. Figures 9-16 graphically displays the data for each microarray before and after mean and median normalization. Microarray #1:
Microarray #2:
Microarray #3:
Microarray #4:
Expression Determination Results Statistical analysis was used to determine the amount of expression for genes involved in the pentose phosphate pathway along with ALD6 (table 4). Table 4. Expression Table of Pentose Phosphate Pathway and other Relevant Genes. Genes were determine to be repressed or induced by having a ratio at least one standard deviation away from the mean of all ratios on the slide. Green indicates induction and yellow indicates repression. The "/" represents lack of data for that particular gene
For zms1Δ, TKL1 had a ratio of -3.31 and ALD6 had a ratio of -0.09, both of which were considered to be repressed by being at least 1 standard deviation away from the average. ZMS1 was the only gene induced with a ratio of 0.25. In zms2Δ only ZMS1 was induced with a ratio of 1.82 and only RPE1 was repressed with a ratio of -5.14. The zms1Δ zms2Δ strain exhibited several genes that were repressed; ALD6 with a ratio of -3.25, SOL4 with a ratio of -1.79, GND1 with -1.98, and TKL1, with a ratio of -3.82. The only gene that was induced was ZMS2 with a ratio of 0.65.
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