Introduction*

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DNA microarray technology, a relatively new tool in the scientific field, can be defined as an analytical instrument used to investigate gene expression of a particular organism’s genome and subsequently specific genes of interest within that genome.  The methodology behind this experimental tool consists of a small membrane or specialized glass slide, which contains samples of several genes arranged in a structured manner.  A microarray works by exploiting the ability of an mRNA molecule to hybridize to the DNA template from which it originated. Utilizing an array which contains an assortment DNA samples allows scientists to determine the expression levels of hundreds to thousands of genes within a cell by measuring the amount of mRNA bound to each site on the array.  With the aid of a computer, the amount of mRNA bound to specific spots on the microarray is precisely measured, generating a profile of the cell’s gene expression1.

All aerobic organisms contain anti-oxidants to protect against the toxicity of oxygen in both the form of enzymatic and non-enzymatic molecules3 (Grabowska 2003).  In order for the anti-oxidants to function, there must be a sufficient supply of NADPH present in the cell.  The enzyme glucose-6-phophate dehydrogenase is the first enzyme in the pentose phosphate pathway and catalyzes the reaction that provides the major source of enzymatic NADPH in the cell3 (Grabowska 2003).  In the yeast Saccharomyces cervisiae, the ZWF1 gene encodes the glucose-6-phopate dehydrogenase.  When this gene is disrupted, it produces a methionine auxotrophy and demonstrates phenotypes suggesting oxidative sensitivity3 (Grabowska 2003).

In a preliminary study, the zwf1∆ mutant strain was permitted to grow by multi-copy suppressors of zwf1∆ aerobically without the presence of methionine, but in the presence of 0.5 mM H2O2 (Slekar unpublished data 2005).  Of the colonies isolated, four suppressors were identified through sequence analysis and sub-cloning.  Among these plasmids, ZMS1 and ZMS2 were identified.  It has been determined that C2H2 zinc-finger-containing proteins are encoded by ZMS1 and ZMS2 and are related to DNA binding transcription factors (Slekar unpublished data 2005). 

 In this specific microarray experiment, a strain of baker’s yeast with a double-knockout mutation, zms1∆zms2∆, will be tested for its relativity to oxidative stress mechanisms and the mutation’s effect(s) on other subsequent gene(s).  The yeast strains were obtained from the lab of another professor of James Madison University, Dr. Kimberly Slekar2.  Based on prior experimentation conducted by Dr. Slekar, it was found that ZMS1 and ZMS2 yeast genes, at a high-copy frequency, effectively suppressed the ZWF1 (a mutant gene with loss of function in oxidative stress environments).  The ZMS1 and ZMS2 genes could potentially be transcription factors, which could increase expression of other anti-oxidative gene over the ZWF1 mutation, according to Dr. Slekar’s research.  In this specific undergraduate microarray experiment, a general hypothesis will be issued: it is the goal of this lab to determine how this double-knockout mutation is genetically expressed, (whether it is overexpressed or underexpressed), utilizing microarray analysis as the source of evidence.

Yeast as a Model Organism:

Yeast are almost as tractable as bacteria and viruses, yet they share many similarities to mammalian cells. The environment in which yeast grow is very easy to regulate, and yeast genes are highly manipulative.  Yeast genes can be knocked out, replaced, made to produce proteins, and added to other chromosomes.4

Saccharomyces Cervisiae:

Biologie. Gilles Bourbonnais: http://ici.cegep-ste-foy.qc.ca/profs/gbourbonnais/index.html

*Picture from : http://plantbio.berkeley.edu