Discussion
Gene
expression patterns were analyzed in two mutant strains of S. cerevisiae
by microarray analysis. Studies by the Slekar lab have determined that
yeast with a ZWF1 knockout (∆zwf1)
exhibit sensitivity to hydrogen peroxide as well as
methionine auxotrophy, indicating the yeasts inability to reduce the reactive
oxygen
species (Slekar, 2008). Two multi-copy suppressors of the zwf1∆
phenotype have been identified by the Slekar lab as the
genes ZMS1 and ZMS2. These two genes encode putative zinc-finger transcription
factors and have been knocked out in a series of three yeast strains;
∆zms1,
∆zms2 and a double knockout
∆zms1∆zms2.
Of all the data generated during microarray analysis, two grids, 7 and 8
containing a total of 836 genes were analyzed using data from three hybridized
microarray slides of the ZMS1 mutant and a single slide of the double mutant
ZMS1/2. It was hypothesized that the there would be significant differential
expression between the ZMS1 and ZMS11/2 mutants, but that there would be
no differential expression between the
∆zms1 triplicates. While this hypothesis did
not turn out to be completely correct, a total of twenty genes out of the 836
analyzed were selected and did meet the criteria of the hypothesis (Tables 4 and 5).
Of the twenty genes selected, six have an unknown gene
name and molecular function (YAL045C, YHL008C, YDR065W, YLR431C, YML005W,
YLR392C). All six of these unknown genes are down-regulated in comparison to the
wild type in the ZMS1 mutant and up regulated in comparison to the wild type in
the ZMS1 ZMS2 double mutant. Three of the genes identified were up regulated in
the ZMS1 mutant and down regulated in the double mutant. Those genes include
VTH2 involved with Golgi to vacuole transport, EXG2 which is part of
glucan 1,3-beta-glucosidase activity,
and SKI3 involved in
translation repressor activity of mRNA catabolism.
The rest of the selected genes were down regulated in the ZMS1 mutant and up
regulated in the double mutant. These genes include CWP1, PAI3, LCB2, GLE1,
RPT4, PHO87, MMM1, SYF1, LEA1, IKI1, and PEX1. Of these ten genes, there did not
appear to be a significant trend in their biological function (Tables 4 and 5).
Even though some information and potential genes of
interest were identified, which met the criteria of the original hypothesis,
inconsistencies within the ZMS1 mutant replicates were obvious despite attempts
at standardization and therefore make the data questionable and potentially
unreliable. Due to the difficulties of working with RNA, the experimental
procedure had several opportunities for error. RNases and time of storage may
have contributed to RNA degradation; moreover, the amount of obtainable RNA for
reverse transcription to the cDNA probe was a limiting factor. Other areas of
experimental error may have included the degradation of the probe and Cy5 (red)
dye due to exposure to light and/or ozone. Observation and quantification of
down-regulated genes within the mutant ZMS1 knockout would be harder to identify
if light or ozone exposure had occurred and may explain why a degree of variance
is present among the replicate ZMS1 mutants array slides. Lastly, the
preparation, hybridization, and wash techniques are subject to various human
errors.
Future experimentation is necessary in
order to obtain more reliable and consistent results. Optimization of
experimental conditions, for instance temperature and salt concentration,
could be altered in order to obtain more consistency throughout the slides.
Although inferences and trends may be drawn from the limited data obtained with
grids 7 and 8 of the four microarray slides analyzed, microarray data is
generally replicated several times and remains inconclusive until
verification of the results has been done using standard molecular techniques
such as RT-PCR. Moreover, the remainder of the grids should be analyzed on each
slide to determine global gene expression, and statistical analysis of the
results should also be carried out with more precision and to a greater degree.