Introduction

Many reactive oxygen species are generated and present in the human body, such as hydrogen peroxide, the superoxide anion, and the hydroxyl radical. They can cause oxidative stress and damage cells by taking away electrons, which damages the DNA, and also cause lipid peroxidation reactions which damage the cell membrane. For this reason, our body’s DNA encodes for many cellular anti-oxidant factors, which are highly conserved, to alleviate this problem. Some examples of these factors are superoxide dismutase, catalase, glutathione peroxidase, glutathione, thioredoxin, and glucose 6-phosphate dehydrogenase (G6PD) .

This last anti-oxidant factor and enzyme, G6PD, is a major source of electrons and combats oxidative stress. G6PD catalyzes an important step in the pentose phosphate pathway, which ultimately provides the cell with NADPH. This NADPH is essential in protecting the cell from oxygen radicals because it is involved in biological reduction reactions. G6PD is encoded by the gene ZWF1. ZWF1 is critical for oxidative stress protection, particularly in Baker’s yeast cells.


Why Yeast?

In this study, Baker’s yeast (Saccharomyces cerevisiae) was used to study oxidative stress protection as offered by ZWF1 in more depth. There are many advantages to working with yeast. S. cerevisiae are ideal to use in such a study because they are simple, single-celled eukaryotic organisms which are easy to maintain and replicate simply (relatively quickly at about 8.5-9.5 hours per cycle1). The entire yeast genome has been sequenced and contains all of the anti-oxidant genes found in Eukaryotes. Human cells and yeast cells are also similar in going through cycles of growth and division.

It has been shown that yeast genes regulating these cycles of growth and division are essentially the same as our genes. Almost everything known about the human cell cycle (including mechanisms of cancer proliferation) was originally learned in yeast. 4   The other advantage is that yeast are very similar to humans, gene-wise. Human DNA can be substituted for the equivalent yeast gene and work just fine.4   This was shown in 1985, when Michael Wigler and his associates at Cold Spring Harbor Laboratory "rescued" a mutant yeast cell that lacked an essential developmental gene, the yeast equivalent of the human ras gene, by inserting the human gene into it. 4   His research proved a significant conservation not only of DNA sequence but also of specific biological function. More than 70 additional human genes have proved functional in repairing various mutations in yeast. 4 According to Stanford researcher David Botstein, "What is true for yeast is also true for human."

            Instructions for virtually all of a yeast cell's functions are clearly “spelled out” in 12 million base pairs of DNA encoding about 6,000 genes (genome-www.stanford.edu/Saccharomyces). This was deciphered in 1996. Since there already exists a gene bank for this organism (their entire genome has already been sequenced), a DNA gene chip necessary for microarray analysis can be used. Biologists enjoy working with yeast because almost everything in yeast's environment can be easily manipulated4. Yeast genes can also be controlled in various efficient ways—knocked out, added to certain chromosomes, replaced with other genes, or made to produce proteins at certain times.
 

      
Cell division cycle of Saccharomyces cerevisiae (left)2 and yeast cells (right)3



Why are microarrays suited for this study?

With the technology of microarrays— 2-dimensional slide-chips containing different molecules (ie, DNA, RNA, protein) onto which molecules are typically spotted and consisting of a medium matching unknown and known DNA samples according to base-pairing rules— one can monitor the interactions of thousands of genes simultaneously. The whole genome can be monitored on a single chip, so that when the organism from which the tissue was extracted is placed in a different environment or under a unique physical/chemical condition, the exact genes involved (higher expression, lower expression) can be identified.
 

Autoantigen microarrays chip   
Slide containing spots of each gene from yeast genome (left)
Slide's spots as they appear to a computer after fluorescent-binding (right)
 

            Particular DNA fragments (cDNA or oligonucleotides) are attached as an array of distinct spots on a specially treated glass microscope slide. Usually slides with 20 nucleotide oligomers are used. This is done by a mechanical robotic spotting process. Two unique probe DNA or mRNA mixtures - the reference and the test sample - are given fluorescent red and green labels, combined in solution, then applied to the array.5 The relative amounts of red and green fluorescence at each spot provide a measurement of the relative numbers of red and green labeled fragments attached at the spot, thereby revealing the relative numbers of fragments in the reference and test samples.5  Cy5 (red) and Cy3 (green) fluorescent dyes are most commonly used in this two-color system. They are most often used in research since they are relatively bright and stable.

            The probe usually consists of red fluorescently labeled mRNA (or a corresponding cDNA produced by in vitro reverse transcription) extracted from a test sample, and a green fluorescently labeled cDNA from a reference sample.5  After these labeled probes are mixed in solution, they are hybridized to the array and unbound probe is washed away. The resulting slide is scanned by a fluorescent imaging system to show red (which corresponds to upregulated genes), green (downregulated genes) and yellow (no differential change in expression) intensity measurements from each spot on the array.

            After being normalized, the ratio of these red and green intensities can give information about the change in mRNA levels between the test and reference populations, essentially showing relative levels of gene expression between the two.

            This procedure is basically like carrying out a lot of Northern blots, but it is much more efficient. That is why it is used more often in examining overall changes in gene expression and figuring out which genes (or similarly grouped genes) are affected by the knocked-out or over-expressed genes. The microarray procedure has not been perfected and is usually repeated at least eight times before publishing. PCR and Northern Blot are often used to double-check the data.

 

Details of the study

In this study, microarray technology was used to compare the mutated form of ZWF1 (referred to in this paper as ΔZWF1) with over-expressed and wild type expression of two genes-- ZMS1 and ZMS2. A yeast colony with mutated ZWF1 cannot produce NADPH; since low levels of  NADPH results in oxidative stress, then oxidative stress gene markers will appear. However, the ZMS1 and ZMS2 genes encode potential zinc-finger transcription factors, and it is thought a cell over-expressing wild type ZMS1 and ZMS2 (referred to as ΔZMS1 and ΔZMS2 in this paper) can fight off oxidative stress even in a cell lacking functional G6PD (due to a mutated ZWF1 gene)—in other words, suppress the ZWF1 mutation.
 


                Relative position of ZWF1 gene on yeast chromosome


            Therefore, we conducted this experiment using microarray analysis to investigate the role gene expression in yeast by comparing the mutant ZMS1 (ΔZMS1) with regular expression of ZMS1 and ZMS2 to the mutant ZWF1 (ΔZWF1) with over-expression of ZMS1 and ZMS2 (ΔZMS1 and ΔZMS2). The results of this experiment will determine the relative expression of genes involved in oxidative stress protection system in yeast, hopefully leading us to identify new anti-oxidant pathways.


ZMS1 and ZMS2 are genes which encode zinc fingers. A zinc finger (shown above) is part of a protein that can bind to DNA.6 

Zinc finger domains typically consist of two antiparallel β sheets, each carrying a cysteine residue, and an α helix carrying two histidine residues.7  The cysteine and histidine residues bind a zinc atom. Many transcription factors (such as Zif268), regulatory proteins, and other proteins that interact with DNA, all contain zinc fingers. These proteins possess amino acid sequences that combine with a zinc ion. They typically interact with the major and minor grooves along the double helix of DNA.7  They come in many shapes and sizes, but share the property of all chelating a zinc ion in their binding domain. They also all have a long alpha helix that inserts into the major groove of DNA, making contact with the bases.7

           

 

LITERATURE CITED

1. http://www.talandic.com/main/wave/cell_cycle.html
            2. http://www.phys.ksu.edu/ gene/a2fg21.gif
            3. http://ww.sb-roscoff.fr/ CyCell/Images/yeast1.jpg 
            4. http://www.hhmi.org/genesweshare/a110.html
            5. http://mgm.duke.edu/genome/dna_micro/core/FAQ.htm
            6. http://www.scripps.edu/newsandviews/e_20010430/barbas1.html
            7. http://en.wikipedia.org/wiki/Zinc_finger

 

 

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