EdwardRyanTalatala Week 9

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Purpose

The purpose of this assignment was to learn how to properly read primary research articles, i.e. how to read the paper critically and how to criticize and review the paper. It also teaches us how to interpret figures in the article, as well as how to present them in open discussion in class.


Journal Club 2 Assignment

Biological Terms

  1. transcriptome: the full range of messenger RNA, or mRNA, molecules expressed by an organism. Can also be used to describe the array of mRNA transcripts produced in a particular cell or tissue type.
  2. chemostat: an experimental apparatus where the chemical environment can be maintained static and nutrient availability can be controlled by the experimenter.
  3. hexose-transporter genes: genes that encode transporters for six-carbon monosaccharides
  4. cell wall mannoproteins: yeast cell wall components that are proteins with large numbers of mannose groups attached; highly antigenic.
  5. ergosterol: the predominant sterol in plasma membranes and secretory vesicles, the fractions with the highest sterol levels
  6. homeoviscous adaptation: a process that allows cell membranes to maintain a liquid-crystalline state at temperatures potentially low enough to cause a membrane to enter the gel state.
  7. hypergeometric distribution: a discrete probability distribution that describes the number of successes in a sequence of n draws from a finite population without replacement.
  8. chromatin immunoprecipitation: identify links between the genome and the proteome by monitoring transcription regulation through histone modification (epigenetics) or transcription factor–DNA binding interactions
  9. cis-regulatory motifs: a site that is bound by a transcription factor(TF) under particular circumstances, and this binding plays a significant role in regulating the transcription initiation rate of the TF-target gene, which is usually located in cis to this TF-target site.
  10. ammonia permeases: a membrane protein that increases the permeability of the plasma membrane to a ammonia, by a process not requiring metabolic energy.

Article Outline

What is the main result presented in this paper?

The results showed that there are several transcriptional responses to low temperature under glucose-limitation and ammonia-limitation. To adapt to low-temperature protein-synthesis capacity, cultures increased transcription of ribosome-biogenesis genes. Transcript levels of environmental stress response genes were reduced at 12°C. Analysis of trehalose-biosynthesis genes and trehalose levels concluded that trehalose is not involved in steady-state low-temperature adaptation.

What is the importance or significance of this work?

This paper concluded that transcriptional responses of S. cerevisiane to low temperature and low specific growth rate, two parameters that are intrinsically linked in batch cultures, can be dissected by the use of chemostat cultures. Studying these differences in chemostat cultures proves to be importance for discriminating different phases in the physiological adaptation to environmental change as there were large changes between transcriptome data and literature data.

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

Previous studies that studied the effects of ESR in S. cerevisiane have shown major discrepancies in data and results. They also wanted to understand the role of trehalose as previous studies have determined it's only used for near freezing temperatures. Currently, there is no clear lear low temperature–specific transcriptional network for Msn2p/Msn4p complex. Finally, the differences in transcriptional response to low-temperature adaptation and acclimation have yet to be studied.

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

They studied growth rate of yeast cell at suboptimal temperatures with either limited carbon or limited nitrogen, focusing on genome-wide transcriptional regulation.


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

prototrophic, haploid S. cerevisiae strain CEN.PK113-7D

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

Yeast cell cultures were grown in medium that was limited by carbon or by nitrogen, but excess amounts of all other growth requirements. The yeast were grown at 12°C and at 30°C in anaerobic chemostat cultures.


What controls did they use?

They used chemostat cultures, which can control the dilution rate (D), medium (f, l h1) and the culture volume (V, l). The control yeast were the ones grown on 30°C with excess essential nutrients.

How many replicates did they perform per treatment or timepoint?

3 replicates per culture

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

They performed microarrays using Affymetrix Genechip for the sampling of cells from chemostats, probe preparation, and hybridization. They used the Agilent 2100 Bioanalyzer to determine RNA quality. They used ACT1 transcript as loading standard for conventional Northern analysis.

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

They used Microsoft Excel for pair-wise comparisons for microarray add-in. Heat-map visualizations of transcript data were generated with Expressionist Analyst version 3.2. They used the web-based software Regulatory Sequence Analysis (RSA) Tools to perform promoter analysis. They used Database for Annotation, Visualization and Integrated Discovery (DAVID) 2006 for statistical assessment of overrepresentation of GO biological processes categories. They used Fisher’s exact test assess overrepresentation of transcription-factor binding sites as defined by chromatin immunoprecipitation (ChIP)-on-chip analysis, employing hypergeometric distribution with a Bonferroni correction and a p-value threshold of 0.01.

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

Genome Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under the series number GSE6190.

Briefly state the result shown in each of the figures and tables, not just the ones you are presenting. (X/Y Axis, How were the measurements made? What trends are shown by the plots and what conclusions can you draw from the data?)

  • Table 1
    • This table shows the physiological characteristics of S. cerevisiae grown in ammonium- and glucose-limited anaerobic chemostat culture at both 12°C and 30°C growth temperatures. The values represent the mean +/- SD of data from three independent steady-state chemostat cultivations. The SD were relatively small due to the fact that these characteristics were fixed constant. Because biomass was similar with the two different temperatures, they determined that temperature does not significantly impact growth.
  • Figure 1
    • This Venn diagram compares and contrasts the genes corresponding between c- and n-limited cultures, where 235 genes of 1065 total genes were in common. N-limited cultures had a total of of 571 genes with 202 up-regulated and 369 down-regulated. C-limited cultures had a total of 259 genes with 123 up-regulated and 136 down-regulated. This highlights the fact that there are numerous significantly different genes corresponding to either c- or n-limited cultures with suboptimal temperatures.
  • Figure 2
    • This heatmap illustrates the transcript level ratio of the 1065 genes in anaerobic c- and n-limited cultures. The x-axis represents the different cultures (c-lim @ 12°C&30°C and n-lim @ 12°C&30°C), while the y-axis depicts the expression of the genes, whether they were induced, repressed, or unchanged. The genes were also clustered by their behavior depending on which gene ontology (GO) terms corresponded to the different clusters, including NCR-responsive (NCR=nitrogen catabolite responsive) genes underlined. These significant NCR target genes shows how increased concentration of the residual limiting nutrients at 12°C resulted in a higher degree of catabolite repression. They determined that genes involved in ribosome biogenesis and assembly showed higher transcript levels at 12°C than at 30°C in both c- and n-limited cultures, as well as the fact that n-limited cultures represented increased gene expression involved in protein synthesis and encoding ribosomal proteins at 12°C.
  • Table 2
    • This table shows the protein and storage carbohydrates contents for the c- and n-limited anaerobic cultures at both 12°C and 30°C growth temperatures. The values represent the mean +/- SD for temperature, biomass dry weight, amount of protein in the cell, nitrogen content, trehalose content, and glycogen levels. In the n-lim cultures, trehalose and glycogen levels were significantly lower at 12°C compared to 30°C; however, cellular proteins and nitrogen content were significantly higher at 12°C than at 30°C.
  • Table 3
    • This table identified the significantly overrepresented cis-regulatory binding motifs in 5�' upstream regions, as well as the significantly overrepresented promoter elements that bind known TF or TF pairs according to ChiP-on-chip analysis in low-temperature up- and down-regulated gene clusters derived from cand n-limited experiments. The researchers claim that the 5�' upstream sequences revealed a clear enrichment of PAC cis-regulatory motif. Moreover, as for the genes that that showed a reduced transcript level at 12°C in the n-limited cultures, the promoter analysis revealed an overrepresentation of STRE elements in the upstream regions.
  • Figure 3
    • This Venn diagram and heat map combination compared chemostat culture expressions at low temperatures to all three batch culture results. The x-axis represents the different results from the different datasets, and the y-axis depicts the expression of the genes, whether they were induced, repressed, or unchanged. A total of 259 genes were in common amongst the batches. 91 genes were commonly upregulated. 48 genes were commonly down-regulated. 120 genes showed uncommon results amongst the three batch experiments.
  • Figure 4
    • These Venn diagrams and heat maps compared this study's chemostat culture expressions at low temperatures to all three batch culture results. For the Venn diagram, the similarities and differences between the chemostat cultures and the previous experiments for each of the upregulated and downregulated sets of genes. The x-axis represents each of the 4 datasets, as well as the time points for each, and the y-axis represents each of the gene's type of expression. The left heat map shows genes that were upregulated, and the right heat map on the right displays genes that were downregulated at low temperatures in c- and n-limited conditions. 11 genes showed a consistent pattern of regulation between all experiments: PIR3, SFK1, YPC1, YEL073C, YNL024C, and YLR225C were upregulated, while PHO84, FUI1, AHA1, FCY2, and YLR413W were downregulated. Their main conclusion of this figure was that comparing datasets with different methods and time periods.
  • Figure 5
    • These Venn diagrams compared genes specifically upregulated or downregulated during acclimation using chemostat cultures or adaptation using previous research datasets to low temperature with the growth rate–dependent genes. There were very few overlaps of genes for both the acclimation and adaptation diagrams, but the adaptations diagrams have more overlap. However, there were only 0.7%, which they determined was negligible.
  • Figure 6
    • These Venn diagrams compared genes specifically upregulated or downregulated during acclimation using chemostat cultures or adaptation using previous research datasets to low temperature with the ESR genes. None of the 4 diagrams had common genes amongst batch studies upregulated genes, batch studies downregulated genes, AND ESR genes. However, these diagrams did have varying overlapping, albeit, none with all three criteria. These results show that there are discrepancies in results amongst the experiments.


How does this work compare with previous studies?

The results of this experiment, which took into consideration acclimation vs. adaptation, found that there are many discrepancies among the chemostat cultures and the previous experiments. The only group of genes that were similarly regulated among the studies were related to lipid metabolism.

What are the important implications of this work?

They discussed that it is not possible to change a parameter without an effect on any others, even using chemostat culture, because of the many discrepancies of results among the datasets studied in this paper. They also found that trehalose is not involved in steady-state low-temperature adaptation, which is not what they were expecting. They found significant differences between transcriptional response to low temperature between long term exposure versus abrupt temperature change. Using the chemostat culture method allows a separation of transcriptional responses to low temperature and low specific growth rate. They emphesized the importance of differentiating phases of physiological adaptation in response to environmental change, as well as the fact that S. cerevisiae response to low temperature is not entirely dependent on changes in transcription.

What future directions should the authors take?

The authors could possibly use the chemostat culture method for environmental stressors of S. cerevisiae.

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?

They did a good job with the figures to demonstrate their comparisons within the study, as well as with the other studies. The discussion of each table and figure were in several places/sections, so I could have been more consolidated. However, the results and their analysis for the results were easy to follow.

Acknowledgments

  • I worked with my homework partner Sahil Patel over text to discuss our figure and to discover the main conclusions of this research paper.

Except for what is noted above, this individual journal entry was completed by me and not copied from another source. EdwardRyanTalatala (talk) 20:49, 27 March 2019 (PDT) EdwardRyanTalatala (talk) 20:31, 3 April 2019 (PDT)

References

Tai, S. L., Daran-Lapujade, P., Walsh, M. C., Pronk, J. T., & Daran, J. M. (2007). Acclimation of Saccharomyces cerevisiae to low temperature: a chemostat-based transcriptome analysis. Molecular Biology of the Cell, 18(12), 5100-5112. DOI: 10.1091/mbc.e07-02-0131

Dahlquist, K. and Fitzpatrick, B. (2019). BIOL388/S19:Week 9. [online] openwetware.org. Available at:Week 9 Assignment Page [Accessed 27 Mar. 2019].

Biology Online. (2018, September 12). Hexose. Retrieved from https://www.biology-online.org/dictionary/Hexose on 27 March 2019.

Biology Online. (2005, October 3). Mannoproteins. Retrieved from https://www.biology-online.org/dictionary/Mannoproteins on 27 March 2019.

Biology Online. (2005, October 3). Permease. Retrieved from https://www.biology-online.org/dictionary/Permease on 27 March 2019.

Science Direct. (2012). Cis-regulatory element. Retrieved from https://www.sciencedirect.com/topics/neuroscience/cis-regulatory-element on 27 March 2019.

Scitable by Nature Education. (2014). Transcriptome. Retrieved from https://www.nature.com/scitable/definition/transcriptome-296 on 27 March 2019.

Science Direct. (2018). Chemostat. Retrieved from https://www.sciencedirect.com/topics/neuroscience/chemostat on 27 March 2019.

Science Direct. (2012). ergosterol. Retrieved from https://www.sciencedirect.com/topics/neuroscience/ergosterol on 27 March 2019.

Science Direct. (2009). Homeoviscous Adaptation. Retrieved from https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/homeoviscous-adaptation on 27 March 2019.

ThermoFisher Scientific. (2009). Chromatin Immunoprecipitation (ChIP) Assay Procedure and Essential Tools. Retrieved from https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/chromatin-ip-chip-assays.html on 27 March 2019.

Math.info. (2007). Hypergeometric Distribution. Retrieved from http://math.info/Probability/Hypergeometric_Distribution/ on 27 March 2019.

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