Knguye66 Week 11

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

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

Purpose

The purpose of the Week 11 assignment is to begin the final project by creating an individual journal with an outline of the journal article assigned (to be studied and analyzed), identify and define 10 new terms, create an annotated bibliography, and lastly, a presentation.

Outline of your journal article

Journal Article: Effects of the Pesticide Thiuram

1. What is the main result presented in this paper?
   • According to the DNA microarray experiment, exposure to pesticide thiuram led to alterations in gene expression in yeast cells.
     • genes involved in detoxification and stress response were induced
     • induction of genes causes other effects:
     • induction of genes in redox and defense against reaction oxygen: oxidative stress
     • induction of genes in DNA repair: positive toxic chemical
2. What is the importance or significance of this work?
   • The purpose of this work was to use DNA microarray technology and treat yeast cells to characterize its toxicity on yeast cells and find a biomarker for candidate for the phenom.
     • In the DNA microarray technology, they screened for genes than can detect pesticide thiuram
     • While pesticide thiuram has been studied before, one of the most important aspects of this study is that they were able to integrate DNA microarray technology to study and analyze a whole genomic response to it (the first ones that did it!).
3. What were the limitations in previous studies that led them to perform this work?
   • Previous experiments studied that thiuram shows direct mutagenicity (ie. salmonella), oral toxicity in rodents, increased "possible" carcinogenesis in humans and rodents, but were unable to identify if mutagenicity was in yeast Sacchoramyces cerevisiae. Other reports have mentioned that thiuram can be used as an alcohol deterrent to inhibit degradation of acetaldehyde, etc. However, like noted in question #2, while pesticide thiuram has been studied before, the significance of this experiment is that all these genomic responses were studied all at once in a comprehensive analysis using DNA microarray technology.
4. How did they treat the yeast cells (what experiment were they doing?)
5. What strain(s) of yeast did they use? Were the strain(s) haploid or diploid?
   • The strain of yeast used was Saccharomyces cerevisiae S288C (alpha SUC2 mal mel gal2 CUP1), used as an indicator strain for the experiment. The strain was not identified as haploid or diploid in the journal article. The yeast growth was investigated and studied at Biomek 2000 Laboratory Automation Workstation (Beck-man Coulter, Inc.)
6. What media did they grow them in? What temperature? What type of incubator? For how long?
   • The yeast cultures were grown in a YPD medium that was diluted and grown overnight, then thiuram was added to grow for an extra 15min-2hrs. Following this, a hybridization solution (20× SSC and 2 μL of 10% SDS were added to 30 μL of cDNA) was applied to the yeast DNA microarray, covered, hybridized for 24-48hrs at 65°C. To characterize the toxicity of thiuram and find biomarker candidates, the team tried to find conditions that lead to strong growth inhibition. Therefore, the yeast cells were cultured with differing concentrations of thiuram and incubated for 24hrs at 25 °C in a YPD medium.
7. What controls did they use?
   • In the "Materials and Methods" section of the journal article, it states that "cDNA made from poly(A)+ RNA of the control was labeled with Cy3 [and Cy5]"(Kitagawa & Takahashi, 2002, pg.3909) in different occasions of the experiment. However, it does not specify what exactly is the control.
8. How many replicates did they perform per treatment or timepoint?
   • The replicates differ depending on the timepoints (ie. 15, 30, 120min). What is important to note is that approximately 6,000 genes were found.
9. What method did they use to prepare the RNA, label it and hybridize it to the microarray?
   • As stated previously in question #6, the yeast cultures were diluted and grown overnight to optical density (OD) in a YPD medium. Cells were later harvested through centrifugation and total RNA was extracted via the hot-phenol method and poly(A)+RNA was purified from 400 micrograms of total RNA with an Oligotex-dt30 mRNA purification kit.
10. What mathematical/statistical method did they use to analyze the data?
    • There were no mathematical or statistical methods used to analyze the data. Much of the information that was used was already found from previous studies and through the MIPS (Munich Information Center for Protein Sequences), a yeast database.
11. Are the data publicly available for download? From which web site?
    • The data is publicly available for download if registered for the website that it is on (ie. this article was published on the American Chemical Society - ACS website). Otherwise, the pdf is not easily downloaded.
12. Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.
    • Figure 1:
      • What do the X and Y axes represent? (A) X-axis: Time, hr; Y-axis: OD (optical density) (B) X-axis: concentration of thiuram, microM; Y-axis: relative growth for 2hrs, %
      • How were the measurements made? (A) yeast cells were inoculated with different concentrations of thiuram in YPD medium at 25°C for 24hrs (B) concentrations of thiuram added to the yeast cells at OD=1 and incubated for 2hrs at 25°C
      • What trends are shown by the plots and what conclusions can you draw from the data? (A) yeast growth was repressed with a concentration of more than 4.4 microM (B) measuring the posttreatment viability of yeast cells that does not cause cellular death, addition of OD=1 decreased concentration of thiuram
    • Figure 2:
      • What do the X and Y axes represent? (A,B,C) X-axis: intensity of Cy3; Y-axis: Intensity of Cy5
      • How were the measurements made? obtained by hybridization of labeled cDNA from thiuram treatment for different minutes (A) 15min. (B) 30 min. (C) 120min. at 25°C
      • What trends are shown by the plots and what conclusions can you draw from the data? (A,B,C) increase exponentially; upper left side is induction of transcription, bottom right side shows repression; prolonged thiuram exposure leads to extensive gene expression alterations (A) 15 minute, 2 induced genes and 23 repressed genes out of 6000 genes (B) 279 induced and 510 repressed (C) 688 induced genes and 1161 repressed genes
    • Figure 3:
      • What do the X and Y axes represent? (B) X-axis: number of induced genes, gray bar: genes induced at 30min and shadowed bar: genes induced at 120min; Y-axis: classification groups of genes induced by thiuram treatment (functional classification of MIPS)
      • How were the measurements made? overview of the genes induced at 30 and 120 minutes
      • What trends are shown by the plots and what conclusions can you draw from the data? induction means transient expression or urgent requirement, thiuram treatments causes an alteration in the expression of membrane factors and increases transport activity;
    • Figure 4:
      • What do the X and Y axes represent? (B) X-axis: number of induced genes, gray bar: genes repressed at 30min and shadowed bar: genes induced at 120min; Y-axis: classification groups of genes induced by thiuram treatment (functional classification of MIPS)
      • How were the measurements made? overview of the genes induced at 30 and 120 minutes
      • What trends are shown by the plots and what conclusions can you draw from the data? genes classified in the groups of “transcription,” “cell growth,” “protein synthesis,” and “carbohydrate metabolism” were more repressed compared to the genes that were induced in these groups; genes related to “cellular communication,” “DNA,” and “lipid etc. metabolism” also showed a higher number of repressed genes compared to those that were induced.
    • Table 1:
      • total of 142 genes induced at both 30min and 120min treatments; the products encoded by the highly induced genes were likely to behave as key factors, oxidative stress by the chemicals likely induced the genes; many of the genes were induced more than 20-fold by the 120 min treatment
    • Table 2:
      • gene expression patterns related to oxidative stress, genes involved in redox cycle and defense against reactive oxygen species; only one enzyme detected as being induced by thiuram treatment; expression of genes in oxidative stress not simple induction, but only dependent on transcriptional factor YAP1
    • Table 3:
      • expression of genes related to mutagenicity; only HSP12 was induced at both time points, induction of others were detected at 30min; induction of SSL2 and PHR1 may show that nucleotide dimer may be increased by thiuram
    • Table 4:
      • microarray for the evaluation of chemical toxicity and selection of biomarkers; each transformant treated with thiuram, GFP was detected in the cell that carried pYKL071W-GFP and pYCR102C-GFP (possible biomarker candidates; detection by promoter activity assay depends on expression intensity
      • 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?
    • Previous studies (as referenced in the article) show the correlation between gene expression patterns (ie. by oxidative stress, carbohydrate metabolism, etc.), however, this study analyzes "all of the above" (if you will), to see if any of this is caused by the induction or repression of thiuram
14. What are the important implications of this work?
    • repressed genes are of less importance than induced genes
    • many genes showed high response in the "carbohydrate metabolism" group, essential for transport reaction of thiuram, inductions of such genes indicate protein denaturation and degradation were induced by thiuram treatment
15. What future directions should the authors take?
    • In the future, the authors can use this microarray technology to investigate the toxicity of other things besides pesticide thirium and other mechanisms.
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?
    • While I believe the paper itself is significant in helping understand the effects of pesticide thirium, it does not specify a lot of things; such as, the control (why we use Cys3 and/or Cys5 to dye the control, what the control is). In the "Results and Discussion" section, however, it does a good job characterizing induced genes by thiuram treatment and creating understandable descriptions for the figures and tables so that the average reader could understand the experiment easier. One of the most important things it lacks on is a timeline/time-point map, the paper seems to jump from one result and discovery to the next, instead of comprising a concise conclusion and explaining the reason why more in-depth. This does, however, makes sense due to the nature of the experiment, using DNA microarray technology to analyze everything - the whole genomic response.

Conclusion

Acknowledgments

This section is in acknowledgement to partner Michael Armas (User:Marmas), as well as, Iliana Crespin (User:Icrespin), and Emma Young (User:eyoung20). I would also like to acknowledge Dr. Dahlquist (User:KDahlquist) for introducing and teaching the topic and direction of this assignment.

"Except for what is noted above, this individual journal entry was completed by me and not copied from another source."

User Page

User:knguye66

Template Page

Template:knguye66

Table of all assignments and journal entries for BIO-367-01

Week	Individual Journal Entry	Shared Journal
Week 1	-	Class Journal Week 1
Week 2	knguye66 Week 2	Class Journal Week 2
Week 3	ILT1/YDR090C Week 3	Class Journal Week 3
Week 4	knguye66 Week 4	Class Journal Week 4
Week 5	DrugCentral Week 5	Class Journal Week 5
Week 6	knguye66 Week 6	Class Journal Week 6
Week 7	knguye66 Week 7	Class Journal Week 7
Week 8	knguye66 Week 8	Class Journal Week 8
Week 9	knguye66 Week 9	Class Journal Week 9
Week 10	knguye66 Week 10	Class Journal Week 10
Week 11	knguye66 Week 11	FunGals
Week 12/13	knguye66 Eyoung20 Week 12/13	FunGals
Week 15	knguye66 Eyoung20 Week 15	Class Journal Week 15

References

• Kitagawa, E., Takahashi, J., Momose, Y., & Iwahashi, H. (2002). Effects of the pesticide thiuram: genome-wide screening of indicator genes by yeast DNA microarray. Environmental science & technology, 36(18), 3908-3915. DOI: 10.1021/es015705v
• Dahlquist, K. (2019, November 7). Week 11. In Wikipedia, Biological Databases. https://xmlpipedb.cs.lmu.edu/biodb/fall2019/index.php/Week_1https://xmlpipedb.cs.lmu.edu/biodb/fall2019/index.php/Week_11