Results:

    This experiment was conducted to try and identify what genes are possibly turned on by ZMS1 and ZMS2. These genes were explored for there possible role in the oxidative stress pathway.  For this experiment wild type genes were compared to genes mutated without the ZMS1 and ZMS2 genes using microarray analysis. To start the experiment yeast cells were cultured and the table above was used to determine the different growth stages of the yeast cells.  The desired growth stage of this experiment is the late log phase, which is the stage where the number of yeast cells is most abundant.    To determine what phase the yeast cells were in, the yeast cells were spectrometry read at 600 nm and they were measured using optical density (O.D.) units.  Based on the previous experiment, the optimal O.D. units for the log phase is approximately one.  The results obtained (Table 1) showed that the O.D. readings of ∆ZMS1 (0.888 O.D.) , ∆ZMS2 (0.81 O.D.), and WT (0.876 O.D.) yeast cell cultures are at the appropriate range for the log phase growth.

Table 1: Optical Density readings for ZMS1, ZMS2, and Wild-type at 600nm. 

Results from the agarose gel electrophoresis of the purified wild-type and ZMS1 (figure 1) show that the purified RNA sample was good based on the brightness and thickness of the line. The purified RNA should be twice as bright at the 28s than the 18s.  The wild-type sample of purified RNA (figure 1) was slightly degraded based on the band size and visibility. However, the lack of visibility could be due to poor pipetting technique. The RNA samples chosen for the making the cDNAs were not only based on the agarose gel results but the concentration (ng/µl) and the purity level from the nanodrop absorbance reading at 260nm.

The purity readings from the nanodrop for ZMS1 and wild-type were determined from absorbance ratio at 260nm and 280 nm and were 2.26 and 2.13, respectively.  The nanodrop reading from ZMS1 showed that the concentration of RNA in the sample was 1247.5 ng/µL and wild type was 1043.5 ng/µL.  The RNA samples chosen for making the cDNAs was the wild-type from our group, which had a concentration of 1043.5 ng/ul of RNA and a purity reading of 2.18 from the absorbance ratio. The ZMS2 sample was chosen from group LLS based on their RNA concentration of 1785.1 ng/ul and the purity from the absorbance ratio of 2.13.   The ZMS1 mutation previously examined was no further used for this experiment because of it low purity and concentration levels.   

After the cDNA was hybridized on the microarray plate it was then sent to Davidson college to be processed. Figure 2 shows the image of the microarray data collected from the class of 2004, slide 104. The microarray from slide 113 for this experiment it did produced any usable data. The microarray data from slide 104 was re-analyzed using ScanAlyzed to align the spots and removed unusable data such as large dust spots and or blobby spots. The data collected here was then further analyzed in Magic tool to identify genes and expression patterns. In Magic tool box graphs (figure 3 and 4) were generated to organize the gene expression patterns. The normalized data allowed for the data to be organized on a common scale and minimized the effect of systematic sources of variation (figure 3).

The results from the un-normalized (Figure 3) data showed that there were 4396 genes that were down regulated and only two genes that were up regulated. After looking at the data and four genes were selected that were consistently down regulated (table 3 & 4). These genes of interest were then examined to see if they contained similar consensus sequences that might possibly code for the ZMS2 transcription factor. These genes were YPL223C, YGR212W, TBL025W, and YBL088C. To first see if these genes had similar consensus sequences the promoter sequences for these genes was looked up under the Yeast Genome site. Figure 5, 6, 7, 8 show there genes with the highlight segments of DNA sequences with the promoter region one thousand base pairs either upstream or downstream.

	These genetic regions were compared using Megaline.  We found two conserved sequences that we believe to be the TATA and CAAT boxes

that are universally conserved in eukaryote gene promoters.  The only possible consensus sequence we found for ZMS2 is TTGGGTT, more research into this 

sequence could prove to be the main binding site for ZMS2.

 Table 2:  Quantifying RNA Concentration. 

Figure 1: Agarose Gel of RNA for Wt and ZMS1

Figure 1:Agarose Gel of RNA from ZMS1 and wild-type.  Lane 4 is the wild-type and Lane 5 is the ZMS1

purified RNA. Lanes 2, 6, 7, and 8 corresponded to other groups ZMS1, ZMS2, and wild-type samples.

The 28s and 18s are the ribosomal RNAs (rRNA).

Figure 2: Microarray data picture

Figure 3: Box plot of red and green pixel data after normalization

Figure 4: Box plot of red and green pixel data before normalization

Table 3: Down regulated genes

Table 4: Up regulated genes

Figure 5: 1 kb promoter sequence for gene YPL223

ATGCGGTGGTCGAAGCTTCTATTACATGCCTGAGGTGCGGGTCTCACCCTTGAGCAGCAA

GTGAGAAATTTTACCCTAATTTCGTCTTCTTTTTTCTTTTTTCTTCATCTGGA

CCTATTATTGGTCATGATGCCACTTTTTACAAACTATTTCATATGTTTAATCTAGTTTAT

TTAGATCTACAGCTGTCAGTTTATACGTTTTTATTAATGTCAGTTTGATTTGATTAGTAG

TGACCTTTTTCGTCGAGGTATGCCGTTTCACGGACCTCCAGATCCAGGTGTATAACAATA

GAATTATGGGAAAACGGGGTGGGAAGGTCGTTGTCTAACTCAAACCTCCTGGAATTAGCG

GGTAAAGTGTCGTTATTGTGCTGGGCAACAACTTTTTTTTACGATTTCTTTTTACCTCTC

ATCTAGGTTAGTGTTAGTTGTAAAAACCCGAGCAGATTGGTGCCTTTTCAAGTGCGTTCT

TACAACCATACTTTTTTATTCTAGAACGTGTAAGCAATTACTTTCTCAAAAAAGCAAACG

CAATAAAGATATGCTACGGATAAGTATTGACTCTATCAAGCAATTCGGTTCCTTTGTGAC

AGGTTATAACAACACTAGTTACCATGCTGCCGGGAGGGCCATAAGAACATCAAGCCTGTA

CAGTACAATGATCTCAGCCAACCCGAGAAGGTGCCTGCATTCTTCGAAACTGCTAAATAA

AGAAGGTCAAGAAGAGGGCTATAATGAACAGCTCATTTCAAAAATGTCTTCACAGAATGG

ATCGAATTCCAGACAGAATGAAAGTGAAGGAAAGAAGGAAGGCAAGGCTTCCTCTGTGAA

GTCGTTGTTGCAACATACTCATAGTCACAGCCACACACATATGCATGACAATCCTTTATT

GTCACTGAACGTACAGCAGATCAAGAAGAATCCTGGCGTAAGAATAACATGGATAGGACT

TGCATCAAATGTGGGGATGGCAGTAGGAAAATTTGTAGGAGGAATCA

Figure 6: 1kb promoter sequence for gene YGR212W

TTGGCTCAGCAAACCCACAGTTTTTGTCAGACGCCACCGACATCGAAAACTTTAACAACG

AGGTGCAAACATTCAGAGCTTCTTGTCCATCGTGTACCCAAGAGTGTGAAACTCATATGA

AGCCAGTAAATATCCCACACTTTAAAGAAGTCATTATCATGTCGACGGTCTGCGATCATT

GTGGTTATAAGTCTAATGAGGTGAAGACCGGTGGTGCCATCCCTGACAAAGGAAGAAGGA

TTACTTTATACTGTGACGATGCAGCTGACTTGTCCCGTGATATTTTGAAATCTGAGACCT

GTAGTATGGTAATTCCTGAATTACATCTTGATATTCAAGAAGGTACATTGGGTGGTAGAT

TCACCACTTTGGAAGGTTTACTAAGACAAGTCTACGAAGAACTAGAATCCCGTATTTTCA

CTCAAACTTCGGATTCCATGGACGAAGCAACGAAAGCCCGCTGGGTAGAATTTTTTGCCA

AGCTAAAGGAGGCCATCGCTGGGAAAGTCAAGTTCACAGTCATTATGGAAGATCCATTGG

CCGGGTCGTACATACAAAATGTCTACGCCCCAGATCCGGATCCAAACATGACTATCGAAG

ATTATGAAAGAACTAAAGAGCAAAATGAAGACCTGGGATTGTCCGATATCAAGGTTGAGT

AACGATCGTTGGCCTCGGTATCACCTCCCCCTTTCCTCTTCCTCTTTACATATATCCTAA

CCACACAAGCACTCATTTGATATGATAATACTTATTCGTTTTTATTCAAATAGATAGCGC

AGTCTTGAAGATTTACCTATATTTTTAAACTTTTGTATAATAGTTGAAATAGATAATACA

GCATTTTTTGGCTCCTGCTTCATATCTTTTTTTTTAGGTTTTTGCTTTATATTCTTTCTT

TTAACTCAACTTGTGCGGAGCAGAGGTAAAGAGGACAACTATAAATGCTGTCAAAACGAA

CAATCTACAGATATTTTTACGAAAAGGAAAAAGCGCAAGA  

Figure 7: 1kb promoter sequence for gene YBL025W

AAGTTTTGAACCAAAAAAAATAAAACCGAAAACAGAGCAACTTTAATTATAGTACGGCAT

ATAAGGAAAACAAATCCAGAATTCAATAAAAGAATACAATACTTTCGAACACATATCTCA

CGCTGATAACACCCTAGCAAGTATGCTTTTCTTCTCCTTTTTCAAGACTTTAGTTGACCA

AGAAGTGGTCGTAGAGGTATGTTCATAATGATTTACATCGGAATTCCCTTTGATACAAGA

AAACTAACGGGTATCGTACATCAATTTTTGAAAAAAGTCAAGTACTAACGTTTGTTTACC

CCTGTTTATTGTGTTTCCACTCAGTTAAAAAACGACATTGAAATAAAAGGTACACTACAA

TCAGTTGACCAATTTTTGAATCTGAAACTAGACAACATATCATGCACAGATGAAAAAAAA

TATCCACACTTGGGTTCCGTAAGGAATATTTTTATAAGAGGTTCAACAGTCAGGTACGTT

TACTTGAATAAGAACATGGTAGATACGAATTTGCTACAAGACGCTACCAGAAGGGAGGTA

ATGACTGAAAGAAAATAAGAATTATGCAGTCTATCCTCATGGCATTTTTTCTCATTAGTT

TTTTACACGAGTATCATTTTAGGATATTGAGATTATATGTATATGTATATATGTAGGCCA

TAAGATGATTCAACGGTTTGTATTAATTATCCGGTTCCGAGCGTTAGGAGAAATAACTTG

GTGTGATATCTGTCAAGTGGTCATCCAAAATTTTTTTGGCCAGAGCGTAAGCTACGTAGA

CTGATGAGGAATTCTTTTTTTTTGCCCCATTTTATCCCAAGAAATTCATTATGAGGTTTT

GTTTTGTTTTACTAAATGCGCTTGATGGAAAAGCGAATGTCATGAAATGCAGATCAACAC

TCCAATAAAAAAGTAAATGTTTTTAAAGAAGTATATCACGCTACAAAAATTCATAAGCAC

TATATTTCGAACGTTAACCTAAATCTCTCCCACTAAGAAA  

Figure 8: 1kb promoter sequence for gene YBL088C

TTGCTATGCAGATGCTTTATACTTCTTTTTTTTGTTTATAGAAATACGAAAAGTAA

TGTACAAACTCAATGTGATGATGTGCCCAAAAATACAATCGCTGATAGTGAAGCTACCAT

TACTGCTATCCCCCAGATGAATAGAGATAAACTTAAGTATTTGAATATTTTTATGCTTGT

CGGTACTCTTTCATTCGTGCCCGTAAATTCTGAGCCAATTAATTTATAACCAAAAAGGCC

TGGCAAAATGAAAGAAATCGACGTAGATCCCGTGGCACCAACTATTGCTAGGACTTTTGC

TAGAGACGTAATTGAAATAGCCAGTAGATATGAGAACAGTAAGATACAAAGGGTGATAAT

ATTGATATGCCGTAAAGACTCACTACGCAGATTTGGCTCTTCGTTGTTTTGTTGGGTTGG

CGCCTCCTGCGGATCTTCACTATTAAAGTTGTCTAATGGAATAAAGCTAGCTCTGTTATC

GTATAACTTACCTTTTCTGAAATTTTCAATGAATATAATTATGTTTTTTACCGATGATCT

GCAAGGATGGCATTGCAATGGAAATGCTAACATAACTAATAGCAGCATTGCTAACCTCCC

GATGGTGGTGGAGATGGAATTCGGGTATAAAGTGAGGATATTTCCTACAATATTCTCACC

AAATGTCATATAACCTGTACCACCAATTATGATGTATAAAAAATAGGCCAACACGATGGC

AAAAATCGGAATCCTCCTGATTACCTTGAAGCTCTTATCCACTTGCTCATTAATTACACT

GAACATATTGTGGTGACAAGTGTAAGCAAACACAAAAATTGGCAATGTAGTCAGGGGAGA

ATGAGACTGAGAATCTCCGTGAGGTACCATAAAATATACTTGCCCTCTCTCTAGCTGATG

CCGATTTACAAAATGGTAAATAATCAAACCAGATAAATATGCGACACTAACAATGGCAAT

CATAGAAGCATATCGTAGAGAATTCAAACTTCTTTTAAAGCATA

Index

Introduction Page

Material and Methods

Discussion