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Introduction

Oxidative stress is believed to be caused by free radicals in the cells.  These free radicals can damage cell components including DNA, proteins, and lipids in the cell membrane.¹  The free radicals include superoxide anions, hydroxyl radicals, and hydrogen peroxide.²  In yeast, several genetic anti-oxidant factors have been identified.  These include:  superoxide dismutase, catalase, glutathione peroxidase, glutathione, thioredoxin, and glucose-6-phosphate dehydrogenase.  In the glucose-6-phosphate dehydrogenase pathway, several NADPs are produced.  The NADPs can attach to a hydrogen ion to form NADPH.  This collecting of the hydrogen ion is one example of free radical accumulation.  The accumulation of hydrogen ions or free radicals by NADP protects the cell from damage.¹

            In order to test genetic pathways employed in oxidative stress, the organism Saccharomyces cerevisiae or baker’s yeast is used.  S. cerevisiae is an important prototype because it is a single-cell eukaryote that can easily be manipulated genetically.  Also, its entire genome has been sequenced and it has many of the similar anti-oxidant genes as all other eukaryotes.¹ 

            In recent research, the genes ZMS1 and ZMS2 were knocked out of S. cerevisiae.  The genes from these yeasts were tested with microarray technology.  ZMS1 gene is a zinc finger transcription factor for respiratory growth in glycerol carbon based nutrients.  It appears to interact with mitochondria and nuclear DNA, but it is unclear the pathway of these genes.³

            In my research, I will be using microarray technology to review the S. cerevisiae genes in ZMS1 and ZMS 2 knockout yeast.  I will be evaluating ten genes across six slides.  Three of the slides will be ZMS1 knockout yeast genes and three slides will be ZMS2 knockout yeast genes.  The ZMS1 knockout slides include slide numbers 13760695, 13760694 and 13760722.  The slides for ZMS2 knockout include 13760725, 13760726 and 13760728.  I will be looking for similar up or down regulation across both types of knockout yeast or differences between each strain of yeast.  The change in regulation may give insight into the pathways of ZMS1 and ZMS2 gene activity.

Methods and Material

Please refer to the web-page Oxidative Stress in Yeast Cells by Heath Davis, William Cannon and Kathy Ware.  All methods and materials were the same.

Results

            The slides were analyzed with Scananlzye and the data was then loaded into Magic Tool.  The Magic Tool program was created by Davidson College.  The following results were developed with Magic Tool. 

            Once the data was loaded into Magic Tool, the data was normalized.  Normalization is needed to control the dye bias; the intensity of the red dye or the possible fading of the red dye.  Figure 1 shows the difference between the pre and post normalization. 

Box plot graph of data prior to normalization

Box plot after normalization

Figure 1:  The data in the first box-plot is before normalization.  The data in the second box plot is after normalization.  Prior to normalization the mean was one to two points below zero.  After normalization, the mean is closer to zero.  The columns are as follows: column one is slide 13760695, column two is slide 13760722, column three is slide 13760694, column four is slide 13760728, column five is slide 13760726 and column six is slide 13760725.  The first three columns contain the data for ZMS1 knockout yeast.  The last three columns contain the data for ZMS2 knockout yeast. 

            Following the normalization process, the expression of the genes is reviewed over two grids for each slide and ten genes were focused.  In Figure 2 a line graph shows the regulation of all genes explored in this research across all six slides.

                   ZMS1                            ZMS1                               ZMS1                                ZMS2                              ZMS2                              ZMS2                 

                13760695               13760722                13760694                  13760728                13760726                    13760725             

Figure 2:  A line-plot showing all 75 genes within two grids reviewed across six slides.  The each line represents the expression of a gene in each slide. 

Next, the data was placed into a format to view each gene separately across all six slides.  The genes were then reviewed for similar expression across all six slides or a different expression across the slides between ZMS1 and ZMS2 yeast.  The genes of focus had a three fold increase either up or down regulated (Figure 3). 

Figure 3:  Magic Tool expression file for the ten genes of interest.  Many of the genes were down regulated across all slides and both the ZMS1 and ZMS2.  However, three genes had an up regulation in the ZMS1 and down regulation in ZMS2.  Green represents down regulation and red represents up regulation.

            The ten genes of interest were found on the GCAT Yeast Gene List found at the following web-page: http://www.bio.davidson.edu/projects/gcat/GCATprotocols.html#yeast.  The ten genes of interest are found in Table 1. 

Table 1:  The first 6 genes and YKL071W were down regulated across all six slides.  Genes YBL051C, YJL216C and YNL062C were up regulated in ZMS1 knockout yeast and down regulated in ZMS2 knockout yeast.⁴

Discussion

            Oxidative stress is caused by free radicals in the cells.  These free radicals can cause damage to DNA, lipids and proteins.  However, certain genetic pathways may help control the number of free radicals in the cell.  In the knockout yeast ZMS1 and ZMS2, it is thought these zinc finger transcription factors control free radicals.  Since these genes are transcription factors, it is of interest what other genes and genetic pathways are affected by the removal of ZMS1 and ZMS2 genes?  The change in the regulation of the genes may mean these genes are part of the ZMS1 and ZMS2 pathways. 

Several of the genes reviewed have a known functions.  Gene CDC13 controls cellular reproduction by controlling the telomeres on the chromosomes.  Therefore, this gene can halt the cell cycle.⁵   the gene JEM1 is connected with the endoplasmic reticulum.  When the cell is under heat stress, the transcription of JEM1 can be stopped.  When it is associated with SCJ1 gene, cell growth will end.⁶ 

However, several of these genes have unknown function.  Many of the genes reviewed have unknown biological and molecular function (YJL152W, YJL073W, YMR209C, YDL204W, YDR161W and YKL071W).  It is possible these genes have a similar function in the cell since they have similar down regulation.⁷  Even though, the RTN2 gene is listed in Table 1 as having an unknown function, recent research suggests this gene has an important biological function when the cell is in a low nitrogen environment.  In a low nitrogen environment, this gene activity is up regulated.   It is down regulated in this research but it could be related to a stress pathway and should be reviewed as a possible oxidative stress gene.⁸  Finally, YKL071W gene has been noted to produce several sequences similar to the A. parasiticusnor-1 protein.  This protein has been related to oxidative stress.  However, it is not found to change expression in the environmental stress of Thiuram.⁹  In this research, the YKL071W gene was found to be down regulated so its expression did not increase in the stress of removal of ZMS1 or ZMS2, but it could be part of the pathways.  These results as well as all results will require further testing.

The results need to be further studied.  This report is only focusing only three slides per knockout gene and microarray tests are run multiple times to obtain reliable results.  Therefore the change in expression needs to be considered across eight or more slides.  Also, dye bias and dye fading due to ozone need to be considered.  Human error affecting the results could included:  not loading enough cDNA for hybridization, hybridization and washing under the wrong temperature, mishandling of the RNase.  Therefore, all results need to be further tested with real time PCR and Northern Blotting techniques to verify results.

Literature Cited

1. Slekar, K.  A Genetic Study of Anti-Oxidant Factors in Yeast.  Lecture 2008 Oct 8. 

2. Cooper, K., Mallory, M., Strich, R.  Oxidative Stress-Induced Destruction of the Yeast C-Type Cyclin Ume3p Requires Phosphatidylinositol-Specific Phospholipase C and the 26S Proteasome.  Molecular and Cellular Biology.  1999:  19(5); 3338-3348.

3. Lu, L., Roberts, G., Oszust, C.  The YJR127C/ZMS1 gene product is involved in glycerol-based respiratory growth of the yeast Saccharomyces cerevisiae.  Current Genetics.  2005:  48; 235-246. 

4. http://www.bio.davidson.edu/projects/gcat/GCATprotocols.html#yeast  Davidson College.  2007.

5. Weber, L., Byers, B.  A RAD9-Dependent Checkpoint Blocks Meiosis of cdc12 Yeast Cells.  Genetics. 1992:  131; 55-63. 

6. Nishikawa, S., Endo, T.  The Yeast JEM1p Is a Dna-J-like Protein of the Endoplasmic Reticulum Membrane Required for Nuclear Fusion.  The Journal of Biological Chemistry.  1997:  272(20); 12889-12892.

7. Rife, T.  Microarray Lecture.  Advance Molecular Biology.  Fall 2008.

8. Mendes-Ferreira, A., del Olmo, M., Garcia-Martinez, J., Jimenez-Marti, E., Leao, C., Mendes-Faia, A., Perez-Ortin, J.  Saccharomyces cerevisiae Signature Genes for Predicting Nitrogen Deficiency during Alcoholic Fermentation.  Applied and Environmental Microbiology.  2007:  73(16); 5363-5369.

9. Kitagawa, E., Takahashi, J., Momose, Y., Iwahashi, H.  Effects of the Pesticide Thiuram:  Genome-wide Screening of Indicator Genes by Yeast DNA Microarray.  Environmental Science and Technology.  2002:  36(18); 3908-3915.