Angela C Abarquez Week 9

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Purpose

The purpose of this assignment is to comprehensively read and evaluate a scientific article while learning about the chemostat-based method of analyzing how yeast acclimate to cold. A presentation of a data table from this article will also be given at the next Journal Club Meeting.

Journal Club 2

The following paper was read and analyzed for Journal Club 2:

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

Defined Biological Terms

  1. Trehalose: A white crystalline disaccharide carbohydrate. It is made of two glucose molecules joined by a α-glycosidic bond (biology-online.org, 2019)
  2. Mannoproteins: Protein components of the yeast cell wall that have many attached mannose groups. They are highly antigenic (biology-onine.org, 2005)
  3. Nucleolar: Of or relating to the nucleolus (biology-online.org, 2017)
  4. Chemostat: A grown vessel where fresh medium is delivered at a constant rate. Cells and spent medium overflow at this same rate (sciencedirect.com, 2010)
  5. Transcriptome: The complete set of transcripts in a cell including their quantity, for a specific developmental stage or physiological condition (Wang et al., 2009)
  6. Hexose: A monosaccharide made of 6 carbon atoms (biology-online.org, 2018)
  7. Biogenesis: The concept that living things only come from other living things (biology-online.org, 2019)
  8. Catabolite: The product formed when complex molecules are broken down into simpler molecules (biology-online.org, 2005)
  9. Cis-regulatory: A noncoding DNA sequence in/near a gene. Although noncoding, it is required for successful expression of the gene and often contains binding sites for transcription factors (nature.com, 2005)
  10. Motif: The smallest group of atoms in a polymer that assemble the other atoms in the chain when influenced by a rotation-translation operator (biology-online, 2008)

Article Outline

Abstract

  • Genome-wide transcriptional responses to cold were studied in steady-state chemostats grown at a fixed growth rate to eliminate indirect effects.
  • Main findings: Transcription of ribosome-biogenesis genes increased while that of environmental stress response genes decreased with cold. Transcriptional change during long-term cold periods differs from the response to a rapid transition to cold.

Introduction

  • Cold temperatures cause yeast to slow their enzyme kinetics and cellular processes.
  • In cold-shock experiments, time of exposure is important as sudden exposure triggers a more rapid and dynamic response while longer exposure can lead to acclimation.
  • Previous studies show an early cold response that occurs within the first 12 hours of exposure to cold and a late cold response that occurs later than 12 hours.
  • There are some discrepancies in the findings of previous studies, such as in the expression of ribosomal protein genes and trehalose accumulation.
  • All previous experiments used batch cultures, which is not ideal for prolonged cold exposure because of the effect on specific growth rate.
  • Chemostat cultures allow more accurate control of specific growth rate and concentrations of the metabolites and substrates remain constant.
  • Relevance of this study: Specific growth rate has a strong impact on genome-wide transcripts and has not been studied using a chemostat culture yet.

Materials and Methods

  • Anaerobic chemostat cultures were used to grow haploid S. cerevisiae at 12 or 30°C with a fixed growth rate of 0.03 h
  • Both glucose-limited and ammonium-limited chemostat cultures were used. A defined synthetic medium limited by either carbon or nitrogen along with an excess of all other growth requirements was used.
  • The time/time points of the experiment were not stated, and 2.0 1 chemostats were used. One Petri dish was grown for each of the four conditions (according to Table 1).
  • Concentrations of glucose and metabolites were measured using liquid chromatography. Trehalose and glycogen were measured using methods described by Parrou and Francois (1997). The glucose release by breakdown of these was measured using the UV method from Roche kit no. 0716251 (Almere, The Netherlands).
  • Methods from Piper et al. (2002) were used to take samples of cells from the chemostats, prepare them, and hybridize them to microarrays. The quality of RNA was determined using Agilent 2100 Bioanalyzer (Wilmington, DE).
  • Three independently cultured replicates were used to derive the results for each growth condition.
  • The following were used to perform statistical analysis on the data:
    • Microsoft Excel
    • Expressionist Analyst version 3.2 (Genedata, Basel, Switzerland) to generate Venn diagrams and heat-maps
    • Regulatory Sequence Analysis (RSA) Tools (van Helden et al., 2000) to conduct promoter analysis
    • http://www.geneontology.org/ (Eilbeck et al., 2005) to assess biological processes
    • Database for Annotation, Visualization and Integrated Discovery (DAVID) 2006 (Huang et al., 2007) to assess overrepresentation of GO terms
    • chromatin immunoprecipitation (ChIP)-on-chip analysis to define overrepresentation of transcription-factor binding sites
    • Fisher's test, using a Bonferroni correction and p<0.01 to assess overrepresentation of transcription-factor binding sites
  • References cited in above methods can be found under the "References" section of the article. Link to article can be found under the "Journal Club 2" header of this page.
  • To compare with other S. cerevisiae low-temperature transcriptome datasets, genes with an average fold difference above 2 were deemed significant.

Results

  • The biomass yields and fermentation rates were similar at 12 and 30°C for both the carbon-limited and nitrogen-limited cultures.
  • Temperature response has a dependency on nutrient availability.
  • The emergency stress response (ESR) occurs when adapting to sudden changes to suboptimal temperatures.
Table/Figure Descriptions
  • Table 1: Mean +/- standard deviation of data from the cultures grown in ammonium-limited and glucose-limited anaerobic chemostat cultures in comparison to the three independent steady-state chemostat cultivations. Growth efficiency does not appear to be affected by temperature.
  • Figure 1: Genes with significant change in expression between 12 and 30°C in the carbon-limited and nitrogen-limited conditions. DNA microarray analysis was used to identify the number of significant genes. There seems to be sensitivity to nutrient limitation.
  • Figure 2: Heat map (left) showing the ratio of transcript level of 1065 differentially expressed genes from the experiment. Labels at the top show the condition of the medium and the temperature. The list of terms belong to GO categories, with NCR-responsive genes underlined.
  • Table 2: Contents of proteins and storage carbohydrates of S. cerevisiae biomass when grown in ammonium-limited and glucose-limited cultures. The different limiting nutrients are shown with the mean +/- standard deviation of the data from the 3 steady-state chemostat cultivations.
  • Table 3: Part A shows the identification of the cis-regulatory binding motifs that are significantly overrepresented in 5' upstream regions. Part B lists overrepresented promoter elements that are known to bind transcription factors in low-temperature gene clusters with a change in regulation. This analysis showed an enrichment of PAC cis-regulatory motifs, which helps in transcriptional regulation of genes that encode ribosomal proteins.
  • Figure 3: Differing expressions in genes of a batch culture adapting to cold. The Venn diagram shows the numbers of genes in each of 3 previous studies. The heat map shows the ratio of 259 genes that are common in all 3 of the datasets. These genes all responded to the cold treatment but in different ways.
  • Figure 4: Comparison between the 259 genes that are common in the 3 batch-culture datasets with those of the present experiment using anaerobic glucose- and ammonium-limited chemostat cultures. The Venn diagrams represent the number of genes common to these two datasets, while the heat map shows expression ratios of the common genes. The genes in brackets consistently regulate transcription in all of the datasets.
  • Figure 5: These Venn diagrams show a comparison of genes with a change in regulation in this study (acclimation) to those that overlap in the 3 previous studies (adaptation). This showed an insignificant overlap between the genes in these two sets.
  • Figure 6: These Venn diagrams compare the genes that are specifically up- or down-regulated in the data set of this study to those that are overlapping in the previous studies. This showed a large overlap between the two sets.

Discussion

  • The specific growth rate can be affected by changes in experimental conditions, which could also affect transcription.
  • Chemostat cultures can be used to analyze transcriptional responses and growth rate more specifically.
  • This method can also be used to focus on acclimation to cold versus adaptation, which is seen in the previous studies that use batch cultures.
  • Acclimation to cold in S. cerevisiae does not only involve changes in transcription. Intracellular metabolite levels can also play a role in changing gene regulation.

Personal Review of Article

Since this is the first study on S. cerevisiae that used chemostat cultures instead of batch cultures, I think a next step could be to use this same method on heat-shock. A comparison of adaptation versus acclimation for heat-shock could yield significant results. Overall, I think the authors supported their conclusion well and were convincing that chemostat cultures should be used more often. The only critique I have is that the methods were not very detailed and the amount of time that the cultures were exposed to cold was not included.

Acknowledgments

My homework partner, Desiree, and I exchanged numbers during class on Tuesday 3/26.

The article reference format from the Week 9 Assignment Page was used to reference the article.

Except for what is noted above, this individual journal entry was completed by me and not copied from another source. Angela C Abarquez (talk) 16:24, 27 March 2019 (PDT)

References

Quick Links

User Page: Angela Abarquez

Course Homepage:BIOL388/S19

Assignments:

Week 1: Instructions and Class Journal

Week 2: Instructions and Class Journal and Individual Journal

Week 3: Instructions and Class Journal and Individual Journal

Week 4/5: Instructions and Class Journal and Individual Journal

Week 6: Instructions and Class Journal and Individual Journal

Week 7/8: Instructions and Class Journal and Individual Journal

Week 9: Instructions and Class Journal and Individual Journal

Week 10: Instructions and Class Journal and Individual Journal

Week 11: Instructions and Class Journal and Individual Journal

Week 12: Instructions and Class Journal and Individual Journal

Week 14/15: Instructions and Individual Journal

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