Results

RNA Isolation

RNA was isolated from wild type yeast that had an optical density at 600nm (O.D.) of 1.33 and mutated ZMS2 yeast that had an O.D. of 1.34. This signifies that both yeast cultures were in mid log-phase and had a concentration between 1x10^7 to 5x10^7 cells/ml. Isolated RNA from both cultures had similar concentrations and A260/A280 purities (Table 1). Wild type RNA had a concentration of 0.112ug/ul and a purity of 2.16. Mutant RNA had a concentration of 0.130 and a purity of 2.21. Ideal A260/A280 purities fall between 1.9 and 2.2. Results from RNA ran on an agarose gel were inconclusive (Figure 1). 28s and 18s bands were not visible on the gel. The only band visible could not be identified.

Table 1. RNA concentrations and purities of wild type and mutant ZMS2 yeast. Both wild type and mutant RNA were isolated from yeast in mid-log phase. 

Figure 1. Agarose gel of RNA isolated from wild type and mutated ZMS2 yeast. 1 ug of RNA was ran on a 1.2% agarose gel at 100v for 30 minutes. Lane 1 was loaded with 1ug of wild type RNA and lane 2 was loaded with mutated ZMS2 RNA.

Microarray

The microarray data was assembled into two data sets. The first data set was assembled using our green fluorescence from our green labeled mutant and the green fluorescence from another lab group dealing with the same mutants wild type (green-green). This was possible because we did a dye reversal of their microarray. This was done again because our red data appeared week at first. The second data set was using our red and green fluorescence from our micro array. Both data sets were analyzed (red-green).

Control Spots

Control/Empty spots were analyzed to determine which genes on the microarray were significantly induced and repressed. Our data came from a scanned microarray slide. An image of a grid on the microarray slide (#2125) can be seen in Figure 2. Gene spots that had a fluorescence intensity greater than one standard deviation greater than the average fluorescence intensity of all control/empty spots on the microarray were considered as a significantly repressed or induced gene. This data is summarized in Table 2. The average fluorescence intensity plus one standard deviation for the control/empty spots for the green ZMS2, red wild type, and green wild type were 1415, 351, and 454 respectively.

Figure 2. Image of grid (1-3) on the scanned microarray slide (#2125). Notice the photobleaching that is evident due to the large color amplification of the green dye and yet still the red dye is still minute.

Figure 3. Shows our microarray without the color amplification. Notice again the dominance of green florescence over the red.

Table 2. Averages and standard deviations of the fluorescence intensities from the control/empty spots of the green labeled ZMS2 and wild type and the red labeled wild type. The average plus one standard deviation was calculated to evaluate which microarray results were significant.

Global Normalization Curve

Global normalization curves were used to determine if photobleaching of the dyes occurred. The slope of the normalization curve can provide evidence for photobleaching. Ideal curves with little to no photobleaching have a slop close to one. Curves far greater or less than one may reveal that photobleaching occurred in one of the dyes. Global normalization curves of green ZMS2 vs. red wild type microarray fluorescence intensities (Figure 4) and green ZMS2 vs. green wild type microarray fluorescence intensities (FIgure 5). The slope of the global normalization curve for the green ZMS2 vs. the red wild type intensities was 0.52. The slope of the global normalization curve for the green ZMS2 vs. the green wild type intensities was 0.005. The line of best fit was placed in order to compare to the 45^o which represents an even balance of red vs. green fluorescence showing a better balanced microarray.

Figure 4. Global normalization curve of green ZMS2 mutant vs. red wild type microarray fluorescence intensities. The linear regression line represents the slope of the average intensities of all the data. The closer the line lies to the 45^o angle the more balanced the green and red data are. Control/empty spots are labeled in blue.

Figure 5. Global normalization curve of green ZMS2 vs. green wild type (obtained from another lab group) microarray fluorescence intensities. The linear regression line represents the slope of the average intensities of all the data. The closer the line lies to the 45^o angle the more balanced the green and red data are. Control/empty spots are labeled in blue.

Repressed Genes

Many genes were significantly repressed in both the green-red microarray and the green-green microarray. Similarities exist between the results of both microarrays (Table 3). The genes which displayed the most repression that were similar between both slides have the ORF name of YJR011C and YMR165C. YJR011C had a negative log2 fold change of 5.672 (green-red) and 6.426 (green-green). YJR011C is an unverified ORF and codes for a hypothetical protein that has an unknown biological process and molecular function. YMR165C had a negative log2 fold change expression of 4.672 (green-red) and 6.229 (green-green). YMR165C (gene name SMP2) codes for phosphatidate phosphohydrolase and is involved in aerobic respiration. Most of the genes that were repressed in the mutant are unverified; however, some knowledge is known about a few of the repressed genes (Table 4). Among the genes of the green-red microarray, the significantly repressed genes with a known biological function are as follows: SMP2, PAM1, YCL073C (nonexistent gene name), COT1, and SPT7. SMP2 as mentioned before is involved in aerobic respiration. PAM1, which had a negative log2 expression of 4.672, is involved in pseudohyphal growth. YCL073C was negatively expressed in both microarrays and codes for a peroxisomal membrane transporter. The most repressed genes with a known function of the green-green microarray were SNF8, AYT1, SMP2, and PFK26. SNF8 displayed a negative log2 expression of 7.276. This gene codes for sucrose non-fermenting protein, which directs proteins toward vacuoles and provides telomere maintenance. AYT1 displayed a negative log2 expression of 6.741. This gene codes for Acetyltransferase, which catalyzes the reaction to produce isotrichodermin from isotrichodermol.

Table 3. Similar repressed genes of mutated ZMS2 yeast from the green-red and green-green microarrays. Similar repression is based on log₂ fold change. Genes are listed by their ORF name. 

Table 4. Most significant repressed genes with a known biological function of mutated ZMS2 yeast from the green-red and green-green microarray. Repression is based on log₂ fold change. Genes are listed from highest to least repressed. Genes that are colored were repressed in both the green-red and green-green microarray.  

Induced Genes

On our microarray there were over a thousand genes that were reported as being induced in the mutant yeast cells. Much of this may be due again to the photo-bleaching of the red dye. In order to make sure our interpretations were as real as possible we made the minimum induction required to be considered real to be fourfold. Again, the level of induction was found by taking the log₂(#green pixels/#red pixels). To simplify these still extensive results further, the most induced genes were looked at for both the green-green and red-green data we gathered. Table X displays the green-green data while Table Y displays the red-green data. In both tables, log₂ values are also included for the other data set as well. For example Table X includes the highest induced genes for the green-green data set but also includes those same genes induction in the red-green data set.

Table 5: Displays the highest induced genes from the green-green data set. This table also displays the inductions of these genes from the red-green data set. Those genes highlighted may have some significance with the oxidation pathway that our knockout mutant has knocked out because the induction is high in both data sets.

Table 6: Displays the highest induced genes from the red-green data set. This table also displays the inductions of these genes from the green-green data set. Those genes highlighted may have some significance with the oxidation pathway that our knockout mutant has knocked out because the induction is high in both data sets.

Notice that only a few of these genes show up in both genes as highly induced. YLR383W (SMC6) shows up as being highly induced in both data sets. This gene deals with DNA repair mechanisms and with the structural maintenance of chromosomes. YNL017C both YCR059C both show significant expression on one microarray, but not the other. They code for proteins involved with vesicle mediat4ed transport and regulation of amino acid metabolism respectively. YKL119C is significant in both slides. This gene codes for a protein that helps in protein complex assembly.

DNA Repair Pathway

1) MEC1  --> signal transducer required for cell cycle arrest and transcriptional responses promoted by DNA damage.       

2) RAD9  --> DNA damage checkpoint protein; activates RAD 53 and chk1p.          

3) RAD53  --> protein kinase required for cell cycle arrest in response to DNA damage.           

4) DDC1  --> DNA damage checkpoint protein; part of PCNA-like complex required for DNA damage response.           

5) RAD24  -->Checkpoint protein, involved in the activation of the DNA damage and meiotic pachytene checkpoints;subunit of a clamp loader that loads Rad17p-Mec3p-Ddc1p onto DNA.         

6) RAD17 --> Checkpoint protein, involved in the activation of the DNA damage and meiotic pachytene checkpoints; with Mec3p and Ddc1p, forms a clamp that is loaded onto partial duplex DNA.           

7) MEC 3  --> DNA damage and meiotic pachytene checkpoint protein; subunit of a heterotrimeric complex (Rad17p-Mec3p-Ddc1p) that forms a sliding clamp, loaded onto partial duplex DNA by a clamp loader complex.

           Table 7: Shows some genes involved in the DNA repair pathway, their known gene reference name, data from our DNA chip and data from our chip + another group's green (mutant) data.

    The data from table 7 shows that, in general, the genes for DNA repair seem to be induced overall. The exceptions to this are RAD53, DDC1, and RAD24 from the other group's data. Possible explanations for this are differences in technique that produce differing data, or may be due to the fact that most people's data is unreliable because of photobleaching effects. This does not necessarily mean the data is useless-- for instance it's interesting to note that that RAD 24 showed over a 4-fold induction in our chip. We used the 4-fold cut-off as a way to weed out inductions/repressions that can't be relied on because of their proximity to the number 1, which signifies no difference in expression between the mutant or wild-type. Therefore an induction of this magnitude could be significant. We expected to see inductions in these genes due to possible free-radical damage incurred by the cell as a result of it's reduced ability to deal with oxidative stress.

Discussion

Methods

Introduction

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