Introduction

DNA microarrays are tools used for analyzing gene expression.  The technique involves the use of a glass slide which contains the sequences of many genes arranged in a particular order.  The microarray works by taking advantage of the ability of RNA, which is reverse transcribed into cDNA, to hybridize to the DNA from which it originated.  Two fluorescent dyes, green and red, are tagged to the cDNA to analyze the results.  A computer scans the microarray slide and a laser reads and records the amount of fluorescence from each probe as seen in Figure 1.  A ratio of the two colors can be analyzed for gene expression.

The Colors of a Microarray

An image depicting the color expression of a microarray.
 Reproduced with permission from the Office of Science Education, the National Institutes of Health.


 

In this schematic:
 
 GREEN represents Control DNA, where either DNA or cDNA derived from normal tissue is hybridized to the target DNA.
 
 RED represents Sample DNA, where either DNA or cDNA is derived from diseased tissue hybridized to the target DNA.
 
 YELLOW represents a combination of Control and Sample DNA, where both hybridized equally to the target DNA.
 
 BLACK represents areas where neither the Control nor Sample DNA hybridized to the target DNA.

 

Figure 1: Explanation of results generated from a laser of a microarray slide.

 Retrieved from http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html

Saccharomyces cerevisiae, commonly referred to as baker’s yeast, is a model organism used to study many cellular processes.  It is a facultative anaerobe that grows fairly quickly, its entire genome has been sequenced, and it is easily genetically manipulated.  Due to its ability to live oxygen rich environments, oxygen radicals may be produced in these yeast cells.  Oxygen radicals can cause damage to the cell and, in humans, have been found to be involved in cancer, cardiovascular disease, and neurodegenerative.  S. cerevisiae must produce anti-oxidants in order to survive in an oxygenic environment.  In humans, Glucose 6-phosphate dehydrogenase (G6PD) is one such cellular anti-oxidant protein.  ZWF1 is a gene in yeast that is homologous to G6PD.  Dr. Kim Slekar’s laboratory studies the role of the ZWF1 gene in S. cerevisiae response to oxidative stress. 

Mutant zwf1∆ strains of S. cerevisiae have been found to be susceptible to oxidative stress.  However, these mutants are able to regain the ability to grow in oxidative environments when the ZMS1 and ZMS2 genes were over expressed (ZMS1++ and ZMS2++ strains).  ZMS1 is known to be a transcription factor that has a role in the cellular response to oxidative stress (Grabowska & Chelstowska 2003).  ZMS2 is known to code for a zinc-finger protein and is thought to be a transcription factor, but this has not been verified. 

The goal of the experiment was to identify the effect of ZMS2 upregulation (ZMS2++ strains) on gene transcription in S. cerevisiae.  RNA was extracted, reverse transcribed into cDNA and used hybridized to the DNA chips for microarray analysis.  It was hypothesized that if ZMS2 is indeed a transcription factors for genes involved in oxidative stress, then the transcription of a small group of genes (up to 30 or 40 genes) would be affected in the ZMS2++ strains compared to wild-type controls.

In order to gain further information from the microarray analysis, individual projects were also performed.  Four genes that were over expressed across 4 different slides were found and the promoter regions of those genes were analyzed.  The most over expressed and under expressed genes on the zms1Δ zms2Δ slide were found and compared to another slide to determine any similarity.  Other microarray protocols were researched to determine if there are alternate ways to complete this experiment to get usable results.   

 

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