Results
RNA Isolation
RNA was isolated from yeast cultures in the mid-log phase with an O.D. at 600nm of 1.340 for the ∆ZMS1 mutant and an O.D. at 600nm of 1.392 for the wild type. The concentration of the wild type RNA was found to be 0.120µg/µL and the concentration of the mutant RNA was found to be 0.272µg/µL. In addition the RNA purity of the wild type sample was 4.97 and the RNA purity of the mutant sample was 2.78. The RNA gel, found here, shows 5 distinct bands for both the mutant (∆ZMS1) and the wild type yeast strain. The three uppermost bands contain yeast DNA that is still present in the sample. The next two bands correspond to 28S rRNA and 18S rRNA respectively. The blurred band at the bottom contains the 5.8S and 5S rRNA as well as tRNA.
Grid one of the microarray slide #13452127 can be found here. Due to the lack of intensity of the Cy5 (red) dye, which was hybridized to the mutant, only the green-wild type data was used from this slide. The mutant data was taken from a second microarray slide (#13452129), which had hybridized Cy3 (green) dye to the mutant.
Normalization
Upon averaging the negative controls, the average value was 0.2631 with a standard deviation of 1.54. From this information, the critical values were concluded to be 4.8831 and -4.3569. Any gene ratios that fell outside of these critical values demonstrated induction (positive) or reduction (negative) of gene expression. Utilizing the yeast genome GOD list, the gene designations and functions were determined (Fig. 3).
Comparing the mutant to the wild type, these nine genes were found to be induced in the ∆ZMS1 mutant: YPL167C, YDL005C, YPL181W, YPL188W, YPL190C, YPL204W, YBR102C, YBR116C, and YBR118W. The ratios for each of these genes can be found here. YPL167C encodes the catalytic subunit of DNA polymerase zeta while YDL005C codes for a subunit of the RNA polymerase II mediator complex. In addition, YPL181W codes for a protein that relieves transcriptional repression, YPL188W encodes a mitochondrial NADH kinase, YPL190C encodes a single strand DNA-binding protein, YPL204W is a casein kinase I isoform, YBR102C codes for an essential protein in spliceosome assembly and exocytosis, YBR116C encodes an unknown protein, and YBR118W encodes the translational elongation factor EF-1α.
On the other hand, the following four genes were found to be reduced ∆ZMS1 mutant: YDL065C, YPL278C, YER089C, and YJR042W. The ratios for each of these genes can be found here. YDL065C encodes a chaperone and import receptor, YPL278C codes for a hypothetical protein, YER089C codes for a type 2C protein phosphatase, and YJR042W encodes a subunit of the nuclear pore complex.
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