Ntesfaio Week 11
Contents
Purpose
Preparation to create my team's home page, Sulfiknights. Practice how to search an assigned primary literature. Prepare and give a journal club presentation.
10 Terms
proteome
the complement of proteins expressed in a cell, tissue, or organism by a genome https://www.merriam-webster.com/dictionary/proteome
glutathione biosynthesis
Protein involved in the synthesis of the tripeptide glutathione https://www.uniprot.org/keywords/KW-0317
glutathionylation
Protein which is posttranslationally modified by the attachment of a glutathione molecule by a disulfide bond. https://www.uniprot.org/keywords/KW-0318
Antimonite
antimonite refers to a salt of antimony(III), such as NaSb(OH)₄ and NaSbO₂, which can be prepared by reacting alkali with antimony trioxide, Sb₂O₃. https://en.wikipedia.org/wiki/Antimonite
thiol-reactive metals
Molecular Probes’ thiol-reactive reagents will react with thiol groups on proteins to give thioether-coupled products. tools.thermofisher.com/content/sfs/manuals/mp00003.pdf
Outline of your journal article
1. What is the main result presented in this paper?
The purpose of this paper was to observe how Saccharomyces cerevisiae responded to arsenite. This was analyzed by quantitative transcriptome, proteome, and sulfur metabolite. Arsenite exposure can be toxic as it affects transcription in genes which is necessary for protein synthesis. Arsenic is highly toxic and is a risk to human health as well as the environment. Arsenic has been known to poison humans, as seen in Bangladesh and West Bengal where a epidemic of arsenic poisoning occurred as it contaminated drinking water. Chronic exposure can cause cardiovascular disease, neurological disorders, and liver injury. It is also associated with cancers of the skin, bladder, liver, and lung. As organisms because more exposed to arsenite there has been an evolution. Sulfur assimilation in yeasts in performed by having sulfate taken up and metabolized through a sulfate assimilation pathway. The sulfide goes through two possible pathways: the methyl cycle or the glutathione cycle. Glutathione (GSH) is a key factor in a cell's defense of metal toxicity. The metals used were sodium arsenite, cadmium chloride, and potassium antimonyl tartrate. Yeast strains were grown at 30 degrees Celsius on YNB mediums. RNA was isolated from the yeast cells that had been exposed to arsenite. For the proteome analysis yeast cells were exposed to arsenite for one hour. The yeast cells went through gel electrophoresis, protein extraction, and gel analysis. Most genes began responding to the arsenite in the first 15 minutes and peaked after 60 minutes. The lower concentration of 0.2mM triggered a faster transcriptional response that the higher concentration while the expression levels were larger at the higher concentrations. Proteome analysis had two-dimensional gel analysis that confirmed increased levels of proteins in the sulfur assimilation and GSH biosynthesis.
2. What is the importance or significance of this work?
Results showed that exposure to sodium arsenite of different concentrations yielded different effects. Cells exposed to 0.2 mM had a moderate effect on growth while those with 1 mM were severly affected.
3. What were the limitations in previous studies that led them to perform this work?
An understanding of how arsenic affects transcription of genes.
4. How did they treat the yeast cells (what experiment were they doing?)
Yeast strains were grown at 30 degrees celsius on YNB medium that were suplemented with autotrophic requirements. Glucose was used as a carbon source. RNA was isolated from the yeast cells. cDNA was cleaned by vacuum-drying, and resuspension in hybridization buffer. The hybridization mix was incubated at 100 degree celsius for 2 minutes then 37 degrees celsius for 30 minutes. Hybridization was for 12-18 hours at 42 degrees celsius.
5. What strain(s) of yeast did they use? Were the strain(s) haploid or diploid?
S. mikate, S. kudriavzevii, S. bayanus, S. kluyveri, and S. castelli
6. What media did they grow them in? What temperature? What type of incubator? For how long?
7. What controls did they use?
8. How many replicates did they perform per treatment or timepoint?
9. What method did they use to prepare the RNA, label it and hybridize it to the microarray?
10. What mathematical/statistical method did they use to analyze the data?
They used what is called a generalized additive model (GAM) to test if hits were spread equally among all genes.
11. Are the data publicly available for download? From which web site?
Yes, when googling this paper the data is included in the figures and can be downloaded as a PDF.
12. Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.
13. What do the X and Y axes represent?
In Figure 2 the x axis shows metabolite concentrations and the y axis showed time in hours. Figure 3 x axis is the amount of 35S in proteins and GSH. With the y axis being the difference in control and the two arsine concentrations.
14. How were the measurements made?
15. What trends are shown by the plots and what conclusions can you draw from the data?
Figure 1 outlines sulfur assimilation and GSH biosynthesis in response to arsenite. Figure 2 shows a kinetic response of sulfur metabolite in response to arsenite. Figure 3 shows sulfate's incorporation in arsenite exposed cells. Figure 4 shows a heat map of changes of genes in sulfur assimilation. Figure 5 shows sulfur/GSH metabolism contributes to arsenite tolerance.
16. How does this work compare with previous studies?
Haugen et. al previously reported on gene expression data that this paper corroborates to. It integrated phenotype expression and transcriptional profiling by mapping the data. 17. What are the important implications of this work?
Yap1p and Met4p control the response of arsenite tolerance acquisition. Metabolites accumulated over time and sulfur eventually would be metabolized.
18. What future directions should the authors take?
19. 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?
Annotated Bibliography
Conclusion
Acknowledgments
My homework partners this week were User:Jnimmers (Quality Assurance), User:Imacarae (Data Analysis), User:Mavila9 (Data Analysis) , and User:Dmadere (Designer). We each split into different roles (as stated in parenthesis) with myself serving as a Quality Assurance representative and as project manager.
