Mavila9 Week 12 Journal

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N/A	Sulfiknights DA Week 14

Purpose

The purpose of this project was to work as a team on | primary literature to demonstrate our understanding of microarray data analysis.

10 Words I Didn't Understand Before

1. Acute promyelocytic leukemia - An aggressive type of acute myeloid leukemia in which there are too many immature blood-forming cells in the blood and bone marrow. It is usually marked by an exchange of parts of chromosomes 15 and 17. Also called APL and promyelocytic leukemia (NCI Dictionary of Cancer Terms, 2008).
2. Aquaglyceroporin - integral membrane proteins that are permeable to glycerol as well as water (Grosell, 2003).
3. Chelation - the combination of a metal ion with a chemical compound, forming a ring (biology-online.org dictionary, 2014).
4. Glutathionylation - the specific post-translational modification of protein cysteine residues by the addition of the tripeptide glutathione, the most abundant and important low-molecular-mass thiol within most cell types (Dalle-Donne, 2009).
5. Auxotrophic - any strain of microorganism (alga, bacterium, or fungus) that differs from the wild‐type by requiring a supply of one or more growth factors (Oxford Dictionary of Biochemistry and Molecular Biology).
6. Orthologue - a gene, protein, or biopolymeric sequence that is evolutionarily related to another by descent from a common ancestor, having diverged as a result of a speciation event (Oxford Dictionary of Biochemistry and Molecular Biology).
7. Dichotomous - a term that describes dividing into two parts or classifications (biology-online.org dictionary, 2014).
8. Antioxidant - any substance that inhibits oxidation, usually because it is preferentially oxidized itself (Lackie, 2007).
9. Glutathione - most abundant non-protein thiol, synthesized in the cytosol, that protects against oxidative stress, and regulates cell proliferation, immune response, apoptosis, and fibrogenesis (Lu, 2012)
10. Ubiquitination - formation of a highly efficient and selective isopeptide bond between a substrate lysine residue and the C-terminus of ubiquitin catalyzed by ubiquitin-protein ligase, or E3 (Pickart, 2001).

Article Outline

1. What is the main result presented in this paper?
   • This paper's main result was that cells responded to cellular arsenite acquisition by stimulating sulfur assimilation or glutathione biosynthesis pathways through the control of transcription factors Yap1p and Met4p. These results are found by transcriptome, proteome, and sulfur metabolite profiling of Saccharomyces cerevisiae's response to arsenite.
2. What is the importance or significance of this work?
   • This work provides further information about the impact of arsenite on the environmental and human health. Due to its causative and curative properties for disease a more full understanding of global and specific responses can help development of medical therapies.
3. What were the limitations in previous studies that led them to perform this work?
   • Previous studies have not shown whether protein glutothionylation occurs in response to metals or not.
4. How did they treat the yeast cells (what experiment were they doing?)
   • Metal sensitivity assays using sodium arsenite, cadmium chloride, and potassium antimonyl tartrate were done. The yeast cells were exposed to different concentrations of arsenite.
5. What strain(s) of yeast did they use? Were the strain(s) haploid or diploid?
   • The following yeast strains were used:
     • W303-1A
     • RW124
     • CC849-1B
     • RW104
     • YPDAHL1166
6. What media did they grow them in? What temperature? What type of incubator? For how long?
   • The yeast was grown at 30 degrees Celsius in minimal YNB medium of 0.67% yeast nitrogen base, auxotrophic requirements, and 2% glucose or on SC medium.
7. What controls did they use?
   • 18s rRNA was used for a control during Northern blot analysis
   • A culture of yeast unexposed to arsenite was used as a control when comparing GSH synthesis following arsenite exposure
8. How many replicates did they perform per treatment or timepoint?
   • The amount of replicates per treatment was not mentioned in the article.
9. What method did they use to prepare the RNA, label it and hybridize it to the microarray?
   • The RNA was isolated, then 20 micrograms of RNA was primed with 3 micrograms of random hexamer and 3 micrograms of anchored oligo(dT)20 primer. Next the RNA was labeled using a reverse transcription reaction with Cy3-dUTP or Cy5-dUTP in 30 microliters. The RNA was then hybridized with 32P-labeled PCR fragments of MET3, MET25, and MET14.
10. What mathematical/statistical method did they use to analyze the data?
    • A generalized additive model with a logic link was used to analyze whether SGD gene hits were spread equally among all genes or overrepresented among regulated genes
    • SGD hits were obtained from searching for genes with the consensus motif present in the promoter and in at least 50% of the available promoters of other species
11. Are the data publicly available for download? From which web site?
    • The data is publicly available at the end of the paper on https://www.physiology.org/doi/full/10.1152/physiolgenomics.00236.2006
12. Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.
    • Figure 2A showed that within the first 30 minutes of exposure the pools of homocysteine, cystathionine, cysteine, gamma-glutamylcysteine, and GSH started to increase and continued to do so with time.
    • 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?

Files

Sulfinights presentation mavila9

References

(2006). auxotroph. In Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. : Oxford University Press. Retrieved 12 Nov. 2019, from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-1807

Biology Online, BWB Marketing, 12 May 2014, https://www.biology-online.org/dictionary

Dalle-Donne, I., Rossi, R., Colombo, G., Giustarini, D., & Milzani, A. (2009). Protein S-glutathionylation: a regulatory device from bacteria to humans. Trends in biochemical sciences, 34(2), 85-96. doi: https://doi.org/10.1016/j.tibs.2008.11.002

Grosell, M., & Bury, N. R. (2003). Biochimica et Biophysica Acta (BBA)/Biomembranes: Preface. Biochimica et Biophysica Acta-Biomembranes, 1618(2). doi: https://doi.org/10.1016/j.bbamem.2015.10.004

Lackie, J. M., & Lackie, J. M. (Eds.). (2007). The dictionary of cell and molecular biology. Retrieved from https://ebookcentral.proquest.com

Lu, S. C. (2013). Glutathione synthesis. Biochimica et Biophysica Acta (BBA)-General Subjects, 1830(5), 3143-3153. doi: https://doi.org/10.1016/j.bbagen.2012.09.008

“NCI Dictionary of Cancer Terms.” National Cancer Institute, 5 Apr. 2018, www.cancer.gov/publications/dictionaries/cancer-terms/.

Pickart, C. M. (2001). Mechanisms underlying ubiquitination. Annual review of biochemistry, 70(1), 503-533. doi: https://doi.org/10.1146/annurev.biochem.70.1.503