Introduction        Methods        Results        Discussion        References        Photos        Individual Project - Karl

Discussion:

Oxidative stress is responsible for substantial cellular damage to DNA, proteins, carbohydrates, lipids, and ion regulatory systems.  Impairments in the oxidative stress response in humans are linked to cardiovascular disease, as well as neurological diseases such Parkinson’s disease and Alzheimer’s disease (Bond and Greenfield, 2007).  It is believed that the products of the ZMS1 gene are zinc fingers that influence the ability of yeast to handle oxidative stress.  Our goal of this microarray experiment was to investigate the sensitivity of the ZMS1 mutant knockout yeast compared to wild type (Baker’s yeast) and identify specific genes that have significant gene expression differences between the mutant ZMS1 knockout strain compared to the wild type.

Using a nanodrop machine, the purity (Abs 260/ Abs 280) of the RNA extracted from the wild type yeast was found to be 10.6 and for the ∆ZMS1 yeast strain it was found to be 2.2.  The purity value of the wild type strain was not within the acceptable purity range (1.8 to 2.2).  The agarose gel picture (Results: Figure 1) shows that the RNA that we extracted from ∆ZMS1 yeast strain and the wild type yeast appeared to be badly degraded.  Because of the gel results, total RNA extracted from another group of the ∆ZMS1 yeast and wild type yeast was used during the reverse transcription PCR (cDNA synthesis) for the construction of the microarray probes.  

Of the ten selected genes from ∆ZMS1 that had consistent expression level changes (at least 5 of the 7 slides), six of the genes have no known molecular function and five of these genes have not been determined as to which cellular components they comprise. Gene YPL051W, an intracellular protein transporter with monomeric GTase activity, was found to be up-regulated.  Gene YDR165W, responsible tRNA methylation and YAL017W, a gene responsible for protein amino acid phosphorylation, were found to be down-regulated.  Most significantly, gene YML120C, a protein involved with the oxidative phosphorylation of NADH to ubiquinone in complex I of the electron transport chain was found to be down-regulated.  This could be a result of the ∆ZMS1’s sensitivity to oxidative stress.  With down-regulated gene expression in a protein associated with electron transfer, the ZMS1 yeast cells may not have been able to efficiently generate ATP to provide energy for all of its metabolic functions.  We did not have evidence of an increase in gene expression for those genes that are known to resist cellular oxidative stress.

Many of the replicates of the class ∆ZMS1 yeast and wild type yeast microarray slides show conflicting expression level differences.  In the future, more microarray replicates of the ∆ZMS1 yeast and wild type yeast are needed to show consistent gene expression level changes that can be correlated to the absence ZMS1 gene products.  Many class microarray slides showed that the dyed probes were not evenly spread across the slides’ surfaces and gave false gene expression levels.  A technique to allow more even spreading of the probes on the slides may need to be developed. 

The Cye3 (green dye) appear lighter in intensity compared to the Cy5 (red dye).  This could have been due to exposure to light and ozone in the present in the room.  Performing the hybridization of the slides in a dark with no detectable ozone could significantly reduce this complication. Optimization of the microarray slide soaking temperature, buffer salt concentrations, and other buffer components are needed to produce consistent slides.  The stringency buffers of the microarray slide washes may need to be adjusted to enhance proper hybridization of the probes to the slide.

Because microarray data can only establish genetic trends and correlational expression patterns among yeast genes, the data collected must verified by the use of molecular biological techniques.  The verification of the microarray results can be performed by molecular methods such as Southern blot (to confirm integrity of probe construction), and RT-PCR (to confirm relative abundance of specific transcripts in samples).

Noble Egekwu - egekwuni@jmu.edu    Karl Gorzelnik - gorzelkv@jmu.edu    Jonathan Baugher - baughejl@jmu.edu