Marmas Week 11

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Effects of the Pesticide Thiuram: Genome-wide Screening of Indicator Genes by Yeast DNA Microarray

Team Information
Project Manager: Michael Armas
Quality Assurance:  Iliana Crespin
Data Analysis: Emma Young, Kaitlyn Nguyen
Coder: Michael Armas

Purpose

The purpose of this assignment is to begin in-depth analysis of the paper assigned for the final project and presentation of BIOL 367-01. The article assigned to this group, FunGals, is titled "Effects of the Pesticide Thiuram: Genome-wide Screening of Indicator Genes by Yeast DNA Microarray." This week, ten unknown terms from the paper will be defined for clarification, and an outline of the article will be posted to summarize the reading.

Ten Terms

1. Thiuram: "The counting and/or measuring of cells in a fluid suspension" (Oxford Dictionary of Biochemistry and Molecular Biology, 2008).
2. Mutagen: "Any physical or chemical agent that is capable of increasing the frequency of mutation above the spontaneous, background level"(Oxford Dictionary of Biochemistry and Molecular Biology, 2008).
3. Disulfiram: "Trivial name for tetraethylthiuram disulfide; bis(diethylthiocarbamoyl) disulfide; a drug used to deter alcohol abuse in the treatment of alcoholism. Alcohol ingestion after disulfiram causes vasomotor disturbances, nausea, vomiting, and even unconsciousness and death. It acts by inhibiting the enzyme acetaldehyde dehydrogenase and hence slows the removal of acetaldehyde. It occurs naturally in the otherwise edible fruit body of the agaric mushroom Coprinus atramentarius. One proprietary name is Antabuse" (Oxford Dictionary of Biochemistry and Molecular Biology, 2008).
4. Erythrocyte: "A mature red blood cell; in mammals it is non‐nucleated and lacks mitochondria. Erythrocytes contain, but are no longer capable of synthesizing, hemoglobin and they function in the transport of oxygen. Mammalian erythrocytes obtain energy from anaerobic glycolysis and also metabolize glucose via the phosphogluconate pathway"(Oxford Dictionary of Biochemistry and Molecular Biology, 2008).
5. Cytometry: "The counting and/or measuring of cells in a fluid suspension"(Oxford Dictionary of Biochemistry and Molecular Biology, 2008).
6. Glutathione: "A tripeptide that is widely distributed in most if not all cells. It acts as a coenzyme for some enzymes and as an antioxidant in the protection of sulfhydryl groups in enzymes and other proteins; it has a specific role in the reduction of hydrogen peroxide and oxidized ascorbate, and it participates in the γ ‐glutamyl cycle. Oxidation links two molecules by a disulfide bond (represented as GSSG). For clarity, glutathione is sometimes termed reduced glutathione (or the reduced form of glutathione). It is involved in the synthesis of certain leukotrienes"(Oxford Dictionary of Biochemistry and Molecular Biology, 2008).
7. Glutathione Dehydrogenase: "An enzyme that catalyses the reduction by two molecules of glutathione of dehydroascorbate to form oxidized glutathione and ascorbate"(Oxford Dictionary of Biochemistry and Molecular Biology, 2008).
8. Base Excision Repair: "One of the intracellular mechanisms for the repair of DNA lesions (single‐strand breaks, damaged bases, etc.). It occurs in the following stages: (1) recognition of the damaged region; (2) removal of the damaged oligonucleotide by two enzymic nucleolytic reactions (excision); (3) synthesis by DNA polymerase of the excised oligonucleotide using the second (intact) DNA strand as template; and (4) covalent joining by DNA ligase of the newly synthesized segment to the existing ends of the originally damaged DNA strand. The process is light‐independent"(Oxford Dictionary of Biochemistry and Molecular Biology, 2008).
9. Transformant: "A bacterial cell that has undergone transformation, i.e. one that contains integrated donor genes that can be detected by plating on media selective for some or all of the donor genes"(Oxford Dictionary of Biochemistry and Molecular Biology, 2008).
10. Oxidative Stress: "A state of metabolic imbalance within cells that favours pro‐oxidant substances (e.g. superoxide, hydrogen peroxide, hypochlorous acid, nitric oxide, peroxynitrite) rather than antioxidants (e.g. glutathione, ascorbic acid) and antioxidant enzyme systems (e.g. superoxide dismutases, catalase). This leads to oxidative damage to all classes of the major biomolecules. Depending on the degree of imbalance, a cell may die or it might survive in a changed state. Such stress can be important in causing a wide variety of degenerative states, including atherosclerosis, ischemia/reperfusion injury in heart and brain, mutagenesis, and chronic inflammatory disease"(Oxford Dictionary of Biochemistry and Molecular Biology, 2008).

Article Outline

1. What is the main result presented in this paper?
   • This paper showed that the DNA microarray data is used to understand toxicity of chemicals in the environment. Specifically, YKL071W, YCR102C, YLR303W, YLL057C were selected for in the resulting microarray data and used for the promoted activity assay. The thiuram treatment directly affected the promoted of these genes. These results mean that this technique can be used for the selection of biomarkers.
2. What is the importance or significance of this work?
   • With the discoveries from the microarray data, these techniques can be used to find biomarkers for thiuram. By characterizing the toxicity in yeast cells treated with thiuram, yeast will serve as a biomarker as its levels of toxicity can be observed to see the presence of thiuram in an environment.
3. What were the limitations in previous studies that led them to perform this work?
   • Previous studies have shown that the thiuram pesticide is a potentially toxic chemical. Other studies have shown the effects of thiuram on yeast, but its toxic effects have not been observed using a DNA microarray experiment.
4. How did they treat the yeast cells (what experiment were they doing?)
   • Saccharomyces cerevisiae S288C was the strain used for analysis of this experiment. Yeast cultures (OD_660 = 1.0) were treated with 300µL of 50mM thiuram. Samples were prepared that differed by the amount of time allowed to grow in the mixture. These times varied from 15 minutes to 2 hours, after which, the cells were harvested using centrifugation. RNA was extracted by hot-phenol method and purified using a Oligotex-dT30 mRNA purification kit.
5. What strain(s) of yeast did they use? Were the strain(s) haploid or diploid?
   • The researchers used Saccaromyces cerevisiae S288C as the experimental strain. According to Engel et al., S288C is a haploid yeast strain.
6. What media did they grow them in? What temperature? What type of incubator? For how long?
   • The yeast cells were grown in a YPD media at 25˚C overnight. For incubation, a Biomek 2000 Laboratory Automation Workstation from Beckman Counter, Inc. was used at 25˚C.
7. What controls did they use?
   • The control used for this experiment was simply not treated with the thiuram pesticide. Additionally, the control was labeled with a Cy3 fluorescent label, while the thiuram-treated samples were labeled with a Cy5 fluorescent label.
8. How many replicates did they perform per treatment or timepoint?
   • The replicates differed in the time treated with thiuram before cell harvesting. The samples were treated for timepoints between 15 minutes and 2 hours in increments of 15 minutes. However, the most discussed results come from the control group (0 minutes), the 30 minute group, and the 120 minute group.
9. What method did they use to prepare the RNA, label it and hybridize it to the microarray?
   • RNA was extracted by using a hot-phenol method, and poly(A)+ RNA was purified from about 400µg of RNA using a Oligotex dT30 mRNA purification kit. At least 4µg of mRNa and a 0.4µM oligo dT primer were denatured at 70˚C for 5 minutes. This produced the labeled cDNA. Hybridiation onto the DNA microarray was carried out at 65˚C for 24 to 48 hours, after which, the microarray slides were washed with wash buffer twice and rincsed with SCC, centrifuged, and dried.
10. What mathematical/statistical method did they use to analyze the data?
    • Mathematical and statistical methods were not used to analyze the data. However, much of the analysis was performed using Munich Information Center for Protein Sequences (MIPS), a yeast database.
11. Are the data publicly available for download? From which web site?
12. Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.
    • What do the X and Y axes represent?
    • How were the measurements made?
    • What trends are shown by the plots and what conclusions can you draw from the data?
13. How does this work compare with previous studies?
14. What are the important implications of this work?
15. What future directions should the authors take?
16. Give a critical evaluation of how well you think the authors supported their conclusions with the data they showed. Are there any major flaws to the paper?