Abstract Introduction Methods Results Discussion References
The result of RNA isolation can be seen in Table 1. Concentration of WT RNA was 0.226 ug/uL while concentration of ZMS2 RNA was 0.756 ug/uL. The purity of the WT and ZMS2 isolations was 2.83 and 2.12 (Table 1). RNA purity gel showed fade RNA bands with little degradation (Figure 2). However, DNA contamination was also observed (Figure 2).
Table 1. Results of RNA isolation from WT and ΔZMS1
|
|
OD260 |
OD280 |
Purity |
Concentration ug/uL |
|
WT |
0.133 |
0.047 |
2.83 |
0.226 |
|
ZMS2 |
0.378 |
0.177 |
2.12 |
0.756 |
|
1 2 |
|
DNA |
|
tRNA |
|
Ribosomal Subunits |
Figure 2. Gel of RNA purity. Lane 1 contains RNA extracted from wildtype yeast strain and lane 2 contains ZMS2Δ RNA. Each sample was run on a 1.2% agarose gel. The RNA isolated shows little to no degradation; however, there is a sign of DNA contamination.
A sample image of the microarray gridding is shown in Figure 3. The image show one of the 16 grids analyzed. Light blue spots represent those that were flagged for background interference.
Figure 3. Image of gridding of 1 grid (out of 16) on slide 104 top. Parameters were as follows: Column spacing was set at 200um, row spacing at 200 um, spot width at 13 and spot height at 13. Light blue spots show spots that were flagged due to back ground interference with signal.
Figure 4 shows the graph of Cy5 signal intensity versus Cy3 signal intensity graphically. The black line represents the relationship between the signal intensities of the two dyes on the microarray. The red line represents a perfect correlation between the two dyes and what is expected. Data was not normalized but the variation was taken into account during analysis.
Figure 4. Graph of log2 expression levels of ΔZMS1 and WT yeast microarray analysis. Black line represents correlation between the Cy5 and Cy3 signals of the two data sets and is described with the equation y=0.5562x + 4.5726. The red line represents a perfect correlation of Cy3 and Cy5 signal and is represented by the equation y=x.
|
1 2 |
|
tRNA |
|
Ribosomal Subunits |
|
DNA |
Figure 5. RNA viability conformation gel. Lane 1 contains wild type RNA and lane 2 contains ZMS2Δ RNA. The originally isolated RNA samples were re-run on a 1% agarose gel to test for possible RNA degradation, which would be a potential cause for the microarray not producing viable results.
The genes that were indentified as being induced in ∆ZMS1 compared to WT expression are seen in Table 2. There expression profiles in each of the trials can be observed in Figure 6.
Table 2. Induced genes in ZMS1 mutant and their gene function.
|
Gene Name |
Accession Number |
Gene Function |
|
YFL026W |
P06842 |
Receptor for alpha-factor pheromone; seven transmembrane-domain GPCR that interacts with both pheromone and a heterotrimeric G protein to initiate the signaling response that leads to mating between haploid a and alpha cells |
|
YLR243W |
Q06543 |
Factor of eukaryotic transcription 5 |
|
YNL145W |
P34166 |
Mating pheromone a-factor, made by a cells; interacts with alpha cells to induce cell cycle arrest and other responses leading to mating; biogenesis involves C-terminal modification, N-terminal proteolysis, and export; also encoded by MFA1 |
|
YOR202W |
P06633 |
Imidazoleglycerol-phosphate dehydratase, catalyzes the sixth step in histidine biosynthesis; mutations cause histidine auxotrophy and sensitivity to Cu, Co, and Ni salts; transcription is regulated by general amino acid control via Gcn4p |
Figure 6. Graph of gene expression of induced genes of ZMS1 generated using MAGIC Tool with a critical value of 0.9408.
The genes that were identified as being repressed in ∆ZMS1 compared to WT expression are seen in Table 3. There expression profiles in each of the trials can be observed in Figure 7.
Table 3. Repressed genes in ZMS1 mutant and their gene function.
Gene Name
Accession Number
Gene Function
YBR116C
P38268
Dubious open reading frame unlikely to encode a protein, based on available experimental and comparative sequence data; partially overlaps the verified gene TKL2
YDR501W
Q04383
Protein required for partitioning of the 2-micron plasmid/ Transcriptional regulatory protein that targets genes with functions important for the G1/S (start) transition, primarily genes involved in DNA synthesis and repair, chromosome segregation, nuclear division and transcription.
YFL014W
P22943
Plasma membrane localized protein that protects membranes from desiccation; induced by heat shock, oxidative stress, osmostress, stationary phase entry, glucose depletion, oleate and alcohol; regulated by the HOG and Ras-Pka pathways
YFR053C
P04806
Hexokinase isoenzyme 1, a cytosolic protein that catalyzes phosphorylation of glucose during glucose metabolism; expression is highest during growth on non-glucose carbon sources; glucose-induced repression involves the hexokinase Hxk2p
YJR004C
P20840
Alpha-agglutinin of alpha-cells, binds to Aga1p during agglutination, N-terminal half is homologous to the immunoglobulin superfamily and contains binding site for a-agglutinin, C-terminal half is highly glycosylated and contains GPI anchor
YLR327C
Q06177
Protein of unknown function that associates with ribosomes
YLR381W
Q6CPF3
Outer kinetochore protein that forms a complex with Mcm16p and Mcm22p; may bind the kinetochore to spindle microtubules
YLR446W
Q06204
Putative protein of unknown function with similarity to hexokinases; transcript is upregulated during sporulation and the unfolded protein response; YLR446W is not an essential gene
YML100W
Q8TGT0
Uncharacterized protein YML100W-A
YML128C
Q03104
Protein of unknown function; mutant is defective in directing meiotic recombination events to homologous chromatids; the authentic, non-tagged protein is detected in highly purified mitochondria and is phosphorylated/ Meiotic sister chromatid recombination protein 1
Figure 7. Graph of gene expression for repressed genes of ZMS generated using MAGIC Tool with a critical value of -4.2196
Results from the data analysis revealed three genes (SAM4, SAM2, MUP1) that were repressed in ZMS1Δ and two genes (SAM4, MUP2) that were repressed in ZMS2Δ (Table 4). Also, there was one gene (SAM2) that was induced in ZMS1, ZMS2 double mutant (Table 4).
Gene Name
ZMS1
ZMS2
ZMS1/ZMS2
Function
MMP1
YLL061W
-1.52697
1.522853
-0.002056531
High-affinity S-methylmethionine permease, required for utilization of S-methylmethionine as a sulfur source; has similarity to S-adenosylmethionine permease Sam3p
SAM4
YPL273W
-0.01489
-2.28673
-1.150813045
S-adenosylmethionine-homocysteine methyltransferase, functions along with Mht1p in the conversion of S-adenosylmethionine (AdoMet) to methionine to control the methionine/AdoMet ratio
NAT2
YGR147C
-1.24858
0.763034
-0.242772912
N alpha-acetyl-transferase, transfers acetyl group from acetyl coenzyme A to the N-terminal methionine residues of proteins
SPE2
YOL052C
-2.57004
-2.0539
-2.31197103
S-adenosylmethionine decarboxylase, required for the biosynthesis of spermidine and spermine; cells lacking Spe2p require spermine or spermidine for growth in the presence of oxygen but not when grown anaerobically
MAP2
YBL091C
-0.74302
-2.09221
-1.417615241
Methionine aminopeptidase, catalyzes the cotranslational removal of N-terminal methionine from nascent polypeptides; function is partially redundant with that of Map1p
MUP3
YHL036W
-1.46455
-5.64386
-3.554202707
Low affinity methionine permease, similar to Mup1p
MAP1
YLR244C
-0.96219
-0.00871
-0.485449408
Methionine aminopeptidase, catalyzes the cotranslational removal of N-terminal methionine from nascent polypeptides; function is partially redundant with that of Map2p
SAM1
YLR180W
-0.6448
-0.644798299
S-adenosylmethionine synthetase, catalyzes transfer of the adenosyl group of ATP to the sulfur atom of methionine; one of two differentially regulated isozymes (Sam1p and Sam2p)
MET6
YER091C
-1.17521
0.970553
-0.102327798
Cobalamin-independent methionine synthase, involved in amino acid biosynthesis; requires a minimum of two glutamates on the methyltetrahydrofolate substrate, similar to bacterial metE homologs
SAM2
YDR502C
0.432106
-0.33
0.051053141
S-adenosylmethionine synthetase, catalyzes transfer of the adenosyl group of ATP to the sulfur atom of methionine; one of two differentially regulated isozymes (Sam1p and Sam2p)
MUP1
YGR055W
-1.26655
-0.8365
-1.051527606
High affinity methionine permease, integral membrane protein with 13 putative membrane-spanning regions; also involved in cysteine uptake
SAM3
YPL274W
-0.42445
-0.424447959
High-affinity S-adenosylmethionine permease, required for utilization of S-adenosylmethionine as a sulfur source; has similarity to S-methylmethionine permease Mmp1p
Table 4. Data of methionine synthesis genes from slide 104 (ZMS1 knockout mutant), slide 7522 (ZMS2 knockout mutant), slide 106 (ZMS1, ZMS2 double-knockout mutant) along with their functions. Genes were determined to to repressed or induced by having final ratio to be at least one standard deviation away from the mean of all ratios on the slide. The green spots indicate genes that were repressed, red spots indicate genes that were induced, yellow spots indicate genes that were neither repressed nor induced and white spots indicate genes for which data was not obtained.