Ymesfin Week 11

Purpose

The purpose of this assignment is to prepare a journal club presentation based off a given article on yeast.

10 Biological Terms

1. homogenization = the process to create a homogenous mixture of a material. (Biology Online, 2019)
2. cyclin = a category of proteins that regulate the cell division cycle of eukaryotes (Oxford Dictionary of Biochemistry and Molecular Biology, 2006)
3. septins = a category of cytoskeletal proteins associated with GTB Binding protein and creating filaments that are associated with membrane dynamics, vesicle trafficking, apoptosis, and cytoskeletal remodeling (A Dictionary of Biomedicine, 2010)
4. trehalose = a disaccharide consisting of 1-1 α-linkages between glucose molecules (Biology Online, 2019)
5. regulon = a system of two genes on different parts of a chromosome that are systematically regulated by the same regulator molecule [(The Dictionary of Cell and Molecular Biology, 2007)]
6. reporter gene = a gene that synthesizes an easily detectible protein and is also upstream of the gene of interest. Using such genes allows researchers to easily monitor which variable affect the expression of the target gene (The Dictionary of Cell and Molecular Biology, 2007)
7. chemostat = the process by which a bacterial culture is kept in its exponential growth phase by monitoring the concentrations of rate limiting nutrients, media, and cells (A Dictionary of Biomedicine, 2010)
8. b-galactosidase = an enzyme encoded by the lacZ gene in the lac operon responsible for the hydrolysis of beta-galactose from the terminal end of sugars (A Dictionary of Biomedicine, 2010)
9. recombinant = a portion of spliced DNA out of the strand by restriction enzymes and reintroduced to different regions by ligases (The Dictionary of Cell and Molecular Biology, 2007)
10. immunofluorescence = a biochemical method with which a an antibody is linked to a fluorochrome in order to visually detect the presence of a particular antigen (A Dictionary of Biomedicine, 2010)

Outline

What is the main result presented in this paper?

The main findings of this article was that short term deprivation of potassium changes the mRNA levels of Saccharomyces cerevisiae, changes sulfur metabolism, induces oxidative stress, adn triggers the retrograde pathway. A repression of genes involved in ribosome synthesis, translation, adn the cell cycle was also observed after the cells were deprived of potassium.

What is the importance or significance of this work?

Given that potassium is an important cation in the cellular functioning of life, the findings of this study support the possibility of other biochemical pathways that necessitate potassium.

What were the limitations in previous studies that led them to perform this work?

While other studies have demonstrated the relationship between potassium concentrations and large cellular functions such as enzyme activation, other pathways and cellular functions that rely on the availability of potassium have not been explored

How did they treat the yeast cells (what experiment were they doing?)

The yeast cells were transferred to YNB based growth medium that lacked potassium and ammonium, and performed a time course for 10min, 20min, 40min, 60min, and 120min. After doing so, they profiled the expression levels of the genes in the yeast.

What strain(s) of yeast did they use? Were the strain(s) haploid or diploid?

Yeast strains YNR055.1, YPC722, YPC723, YPC724, BY4741, BYT1, BYT2, BYT12, BY4741 rtg2, BY4741 rtg3, BY4741 fzo1, W303-1A, DBY746 were used in this study. Information regarding the ploidy of the yeast was not given.

What media did they grow them in? What temperature? What type of incubator? For how long?

The yeast were grown in translucent potassium-free YNB media at 28°C for 10min, 20min, 40min, 60min,and 120min. Information regarding the incubator was not provided.

What controls did they use?

The study used wild-type strain BY4741 cultured in Translucent media with 50mM KCl as a control.

How many replicates did they perform per treatment or timepoint?

Four replicates were performed per timepoint.

What method did they use to prepare the RNA, label it and hybridize it to the microarray?

Ribo Pure™-Yeast kit was used to extract the RNA. No information was provided regarding how they labeled and hybridized it to the microarray.

What mathematical/statistical method did they use to analyze the data?

GEPAS v3.1 was used to process the microarray data.

Are the data publicly available for download? From which web site?

The data is publically available for downloard from Society for Applied Microbiology

Briefly state the result shown in each of the figures and tables, not just the ones you are presenting.

Fig. 1: Microarray Analysis-The majority of the genes were induced 60-120min after the cells were transferred to potassium free media. X Axis-timepoints; Y Axis-genes

Fig. 2: The genes involved in the creation of MET and SAM that were induced by potassium deprivation were not induced in the trk1 adn trk2 cells.

• 2A: Model demonstrating the sulfur uptake and how Met/Cys are metabolized.
• 2B: Graph depicting relative mRNA concentrations of genes from microarray (X Axis: mRNA change (log2); Y Axis: gene names)
• 2C: Relative concentration of Met/Cys after treatment (X Axis: time (min); Y Axis: Met/Cys (microM)

Fig. 3: Potassium starvation induced genes associated with oxidative stress.

• 3A: Graph demonstrating the change in expression levels of oxidative-stress response genes after potassium starvation. (Y Axis: log2 of change in expression levels; X Axis: Time (min))
• 3B: Images of fluorescent cells demonstrating relative amounts of ROS in each cell after potassium starvation.
• 3C: Graph of the relative concentrations of the oxidized and reduced forms of glutathione after potassium starvation in the cells. (X Axis: Glutathione (mM); X Axis: Time (min))

Fig. 4: This demonstrates the effect of methylglyoxal production as well as trehalose metabolism from potassium starvation.

• 4A: Model depicting how methylglyoxal is generated and degraded and how trehalose is metabolized
• 4B: X Axis- time (min) Y Axis- Fold change in expression shows the change in expression specifically for genes related to trehalose metabolism and methylglyoxal. Measurements were made through the microarray data. (X Axis: Time (min); Y Axis: log2 of change in expression levels)
• 4C: (x axis- time in min and y axis- methylglyoxal concentration) showing MG levels remaining higher after 20 minutes potentially linked to genes induced in trehalose metabolism. (X Axis: Methylglyoxal(microM); X Axis: Time (min))

Fig. 5: Potassium starvation triggers the retrograde response.

• 5A: Graph demonstrating the change in expression levels of retrograde pathway genes after potassium starvation. (Y Axis: log2 of change in expression levels; X Axis: Time (min))
• 5B: Mitochondrial morphology before and after treatment.
• 5C: Graph depicting the relative concentration of intracellular ammonium after treatment. (X Axis: Time (min); Y Axis: mol NH4+/mg)
• 5D: Graph depicting the relative concentration of intracellular ammonium after treatment. (X Axis: Time (min); Y Axis: mol NH4+/mg)

Fig. 6: Potassium Starvation changes cyclin levels

• 6A: Graph demonstrating the change in expression levels of retrograde pathway genes after potassium starvation. (X Axis: time (min); Y Axis: log2 of change in expression levels)
• 6B: Images depicting relative abundance of cyclin mRNA
• 6C: Images depicting relative abundance of cyclin protein

Fig. 7: Potassium starvation decreases septin ring formation

• 7A: Graph demonstrating the change in expression levels of septin ring fomration genes after potassium starvation. (X Axis: time (min); Y Axis: log2 of change in expression levels)
• 7B: Images depicting the localization of Cdc11-GFP during treatment.
• 7C: Images depicting relative abundance of Cdc11 protein
• 7D: Images depicting the localization of Cdc11 protein

How does this work compare with previous studies?

Previous studies have not analyzed the relationship between yeast and potassium concentrations. The potassium free YNB media used in this experiment was not common in many previous studies, and thus added a layer of significance to the implications of this study.

What are the important implications of this work?

The findings of this study demonstrate the relationship between potassium and cellular functions within yeast.

What future directions should the authors take?

The researchers should analyze how the starvation of other cations that have a relationship with potassium, such as sodium, can affect gene expression levels.

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 authors supported each of the main points established in the abstract well by using every figure to corroborate the major results and findings of the treatment. However, I think the authors could have done a better job of introducing major concepts and ideas better. I found myself looking at other sources to understand what the retrograde pathway, oxidative stress, septins, and cyclins were. More information should be added to the introduction to ensure that amateur scientists can understand the content well.

Annotated Bibliography

Keyword Search

1. PubMed
   • What original keyword(s) did you use? How many results did you get?
     • The keywords used in this search were "yeast" (282,936 Results), "potassium" (259,040 Results), and "potassium deprivation" (1,452 Results).
   • Which terms in which combinations were most useful to narrow down the search? How many results did you get after narrowing the search?
     • The most useful combination was "yeast potassium starvation" (13 Results).
2. Google Scholar
   • What original keyword(s) did you use? How many results did you get?
     • The keywords used in this search were "yeast" (315,0000 Results), "potassium" (4,330,000 Results), and "potassium deprivation" (182,000 Results).
   • Which terms in which combinations were most useful to narrow down the search? How many results did you get after narrowing the search?
     • The most useful combination was "yeast potassium starvation" (31,300 Results).
3. Web of Science
   • What original keyword(s) did you use? How many results did you get?
     • The keywords used in this search were "yeast" (272,911 Results), "potassium" (270,927 Results), and "potassium deprivation" (1,025 Results).
   • Which terms in which combinations were most useful to narrow down the search? How many results did you get after narrowing the search?
     • The most useful combination was "yeast potassium starvation" (13 Results).

Advanced Search

1. PubMed
   • What original keyword(s) did you use? How many results did you get?
     • The keywords used in this search were "yeast" (172,332 Results), "potassium" (137,623 Results), and "potassium deprivation" (143 Results).
   • Which terms in which combinations were most useful to narrow down the search? How many results did you get after narrowing the search?
     • The most useful combination was "yeast potassium starvation" (3 Results).
2. Google Scholar
   • What original keyword(s) did you use? How many results did you get?
     • The keywords used in this search were "yeast" (224,000 Results), "potassium" (240,000 Results), and "potassium deprivation" (154 Results).
   • Which terms in which combinations were most useful to narrow down the search? How many results did you get after narrowing the search?
     • The most useful combination was "yeast potassium" (281 Results).
3. Web of Science
   • What original keyword(s) did you use? How many results did you get?
     • The keywords used in this search were "yeast" (89,058 Results), "potassium" (93,444 Results), and "potassium deprivation" (103 Results).
   • Which terms in which combinations were most useful to narrow down the search? How many results did you get after narrowing the search?
     • The most useful combination was "yeast potassium" (195 Results).

Reflect

1. What impact does the choice of keywords have on your results?
2. What are the advantages and disadvantages of each of the three databases (Pubmed, Google Scholar, Web of Science)?

Citations

1. Hess, D. C., Lu, W., Rabinowitz, J. D., & Botstein, D. (2006). Ammonium toxicity and potassium limitation in yeast. PLoS biology, 4(11), e351. DOI: 10.1371/journal.pbio.0040351
2. Navarrete, C., Petrezsélyová, S., Barreto, L., Martínez, J. L., Zahrádka, J., Ariño, J., ... & Ramos, J. (2010). Lack of main K+ uptake systems in Saccharomyces cerevisiae cells affects yeast performance in both potassium-sufficient and potassium-limiting conditions. FEMS yeast research, 10(5), 508-517. DOI: DOI:10.1111/j.1567-1364.2010.00630.x

Data/Files

Short Term Response of Yeast to Potassium Starvation

Short Term Response of Yeast to Potassium Starvation: Supplemental Information

Acknowledgements

Professor: Dr. Dahlquist

Homework Partners: Christina Dominguez, Jonar Cowan, David Ramirez, and Mihir Samdarshi

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

Ymesfin (talk) 20:21, 13 November 2019 (PST)

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.

Biology Online. (2019). Retrieved November 7, 2019 from https://biology-online.org/

Blackshaw, S. E., & Lackie, J. M. (2007). The Dictionary of Cell and Molecular biology. Amsterdam: Academic Press.

Lackie, J., & OCallaghan, C. (2010). A Dictionary of Biomedicine. Oxford: Oxford University Press.

LMU BioDB 2019. (2019). Week 11. Retrieved November 7, 2019, from https://xmlpipedb.cs.lmu.edu/biodb/fall2019/index.php/Week_11

Oxford University Press. (2006). Oxford dictionary of biochemistry and molecular biology. Oxford.

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