Dramir36 Week 11

Purpose

• To learn how to search the primary literature.
• To prepare and give a journal club presentation.

10 definitions of biological terms

• retrograde pathway: The mechanism of moving backward or against the usual direction of flow. Biology Dictionary (2019)
• septin: A protein involved in cytokinesis and also functions in maintaining cell morphology Biology Dictionary (2019)
• β‐galactosidase: One of a class of enzymes which cuts the glycosidic bond between the sugar galactose and another sugar molecule (of a different type than galactose). Biology Dictionary (2019)
• oxidoreductase: An enzyme that catalyzes the transfer of electrons from an electron donor (reductant) to an electron acceptor (oxidant) in a redox reaction Biology Dictionary (2019)
• trehalose: A disaccharide made up of two α-glucose molecules, and serves as an energy source in certain fungi, bacteria, plants, and invertebrates Biology Dictionary (2019)
• supernatant: The soluble liquid fraction of a sample after centrifugation or precipitation of insoluble solids. Biology Dictionary (2019)
• biosynthesis: The production of a complex chemical compound from simpler precursors in a living organism, usually involving enzymes (to catalyze the reaction) and energy source (such as ATP) Biology Dictionary (2019)
• assimilation: The act of absorption or incorporation into the biological tissue. Biology Dictionary (2019)
• isoform: Any of the proteins with the same function and similar amino acid sequence, encoded by different genes (or by RNA transcript). Biology Dictionary (2019)
• attenuate: Tapering gradually. Biology Dictionary (2019)

Outline of Journal Article

1. 1. What is the main result presented in this paper? The main result is that there was a massive alteration in the expression profile shortly after potassium was removed from the medium. The lack of potassium drastically alters the sulfur metabolism, triggers an oxidative stress response, and activates the mitochondrial retrograde pathway. Another main finding was that after a short time without potassium, there were genes that became repressed, including genes that are involved in ribosome synthesis, translation, and the cell cycle.
   2. What is the importance or significance of this work? This research project is important because it shows how when Saccharomyces cerevisiae has a short-term potassium deprivation, there are impacts in the mRNA level of over one thousand genes.
   3. What were the limitations in previous studies that led them to perform this work? There wasn’t much insight of possible cellular functions that require potassium and the short-term effects on the gene expression profile when there is a deprivation from sudden depletion of potassium in the environment.
   4. How did they treat the yeast cells (what experiment were they doing?) Media that contains only traces of potassium and ammonium as nitrogen source, simulated an absence of potassium in the environment. Once the yeast has been incubated at different time points of a potassium-absent environment, RNA purification, cDNA synthesis and DNA microarray was conducted. RT-PCR assays were also conducted to see how the mutants and wild-type reacted to an increase or no increase of KCl.
   5. What strain(s) of yeast did they use? Were the strain(s) haploid or diploid? Wild-type S. cerevisiae had four main strains that were used in the work. The main strain is BY4741. The other three strains were BY4741 without trk1, without trk2, and without both trk1 and trk2. All of the strains used in the work are haploid.
   6. What media did they grow them in? What temperature? What type of incubator? For how long? Recently developed YNB-based growth media was used to treat the yeast cells at 28 degrees Celsius for 10, 20, 40, 60, and 120 min by rapid filtration from four biological replicates. The control contained sufficient potassium, whereas the other media contained only traces of potassium and ammonium. The DH5ɑ cells were grown at 37 degrees Celsius in Luria-Bertani (LB) broth. There was no information given about the incubator that was used.
   7. What controls did they use? The control used was the wild-type strain BY4741 in media that contained 50 ml of KCl.
   8. How many replicates did they perform per treatment or timepoint? There were four biological replicates performed per timepoint.
   9. What method did they use to prepare the RNA, label it and hybridize it to the microarray? The total RNA was extracted by using the Ribo Pure-Yeast kit (Ambion) following the manufacture’s instructions. RNA quality was assessed by electrophoresis in denaturing 0.8% agarose gels and quantified by measuring the A260 in a BioPhotometer (Eppendorf).
   10. What mathematical/statistical method did they use to analyze the data? A given gene was considered to be induced when the minus/ plus potassium signal ratio was equal or higher than 2.0-fold, whereas it was considered to be repressed when this signal was equal or less than 0.50-fold. The GEPAS server (v3.1) was used to pre-process the data.
   11. Are the data publicly available for download? From which web site? The Society for Applied Microbiology website offers the data, which is publicly available, and is located in the article’s “Supporting Information” section.
   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? Table 1: The majority of the genes were induced and repressed 60-120 min after the cells were transferred to potassium free media. Fig. 1: The majority of the genes were induced and repressed 60-120 min after the cells were transferred to potassium free media. Fig. 2: 2A: Potassium starvation decreases sulfuric amino acid metabolism. 2B: Graph depicting relative concentrations of mRNA for genes associated with Met/Cys metabolism. 2C: The concentration of Met/Cys amino acids decreases with potassium treatment Fig. 3: 3A: Oxidative stress response genes are induced by potassium deprivation. 3B: ROS formation causes oxidation when switch to K+-free medium. 3C: Potassium starvation- decline in GSH and increase in GSSG. Fig. 4: 4A: Methylglyoxal increases with trehalose Metabolism. 4B: Genes involved in trehalose synthesis show an increase in expression. 4C: Genes in MG detoxification enhanced without potassium. Fig. 5: 5A: Potassium starvation leads to a strong increase in expression of CIT2 & DLD3 genes central to the retrograde response. 5B: Mitochondrial morphology was normal, and not the cause of the retrograde response. 5C: Potassium starvation leads to an accumulation of ammonium in the cell, and may be one cause of the retrograde response. 5D: Cells without Trk1 do not show significant ammonium accumulation. Fig. 6: 6A: Expression of genes controlling cyclins exhibit a varied response to K+ starvation. Fig. 7: 7A: Lack of potassium in yeast destabilizes the septin ring. 7B: CDC11-GFP localization is affected during K+ starvation. 7C: CDC11 protein levels are constant during starvation. 7D: CDC11 not properly localized in cells grown 60 min
   13. How does this work compare with previous studies? This work demonstrates that lack of potassium actually causes a fast and persistent increase in intracellular ammonium concentration.
   14. What are the important implications of this work? There is a significant amount of change in cells occur even during the retention of more than necessary potassium.
   15. What future directions should the authors take? The authors could separate the methods into their own research projects to prevent intervention or confusion with implementing new methods into the same project.
   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? The only problem that I found with reading the results and the article in general is that, the authors inserted new techniques or analysis without much explanation, or the insertion of a new analysis method was not placed in the right spot of the article.

References

• Barreto, L., Canadell, D., Valverde-Saubí, D., Casamayor, A., & Ariño, J. (2012). The short-term response of yeast to potassium starvation. Environmental Microbiology, 14(11), 3026–3042. doi: 10.1111/j.1462-2920.2012.02887.x, https://sfamjournals.onlinelibrary.wiley.com/doi/full/10.1111/j.1462-2920.2012.02887.x
• Biology Online. (2019). Biology Dictionary. https://www.biology-online.org/dictionary/Main_Page