Alison S King Week 9

Purpose

The purpose is to read more literature about the transcriptional response to cold in yeast and analyze the results of the paper. We also want to be able to critically analyze the effectiveness of the study and the journal and present one of the figures of the paper in class.

Biological Terms and Definitions

chemostat
- a device for the continuous culture of bacterial (and other) cells. Growth occurs in an aerated fermenter vessel and its rate is controlled by the rate of addition of fresh nutrient from a reservoir (i.e. the dilution rate); this in turn controls the rate of removal of cells (and culture medium). In a chemostat, as opposed to an auxostat, the dilution rate is constant. At equilibrium, the rate of production of new cells by multiplication is equal to the rate of removal of grown cells (Cammack et al. 2008)
trehalose
- α‐d‐glucopyranosyl‐α‐d‐glucopyranoside; α,α‐trehalose; a nonreducing disaccharide, also known as mushroom sugar or mycose, that acts as a reserve carbohydrate in certain fungi (especially yeasts), algae, and lichens. It is cleaved by trehalase but not by most other α‐glucosidases. It is a component of cord factors, which are a mixture of trehalose 6,6′‐mycolates (see mycolic acid). α‐d‐Glucopyranosyl‐β‐d‐glucopyranoside (α,β‐trehalose) and β‐d‐glucopyranosyl‐β‐d‐glucopyranoside (β,β‐trehalose) occur naturally, but only very rarely. The carbohydrate is a compatible solute that acts as a cryoprotectant in many microorganisms, and is used industrially as a preservative in foods and pharmaceuticals (Cammack et al. 2008)
diurnal
- occurring during the day.
- repeating every day; having a periodicity of approximately 24 hours. Compare circadian. (Cammack et al. 2008)
prototrophic
- strains that have the same nutritional requirements as the wild-type strain. (Biology Online Dictionary. (2005). Accessed from: https://www.biology-online.org/dictionary/Permease)
upstream
- in or towards positions in a DNA molecule lying in the 5′ direction relative to the start of transcription of a gene. (Cammack et al. 2008)
motif
- (in relation to protein structure) a locally ordered region within the core of a protein molecule, formed by 3‐D interaction between two or three segments of the secondary structure (α‐helix and/or β‐strand) that are near one another along the polypeptide chain. The most important types are: (αα), (αβ), (βββ), and (βαβ). See also domain.
- (in relation to protein sequences or sequence alignments) a contiguous, conserved region, typically 10–20 amino acids in length, usually denoting a key structural or functional unit within a protein, or group of proteins, and often used to diagnose related sequences using pattern‐recognition methods (such as fingerprinting). Alternative name block. (Cammack et al. 2008)
fermentation
- (or ferment) the anaerobic decomposition of chemical substances, brought about by ferments (def. 1), resulting in the production of simpler substances and, often, of energy; an instance of this, especially the anaerobic breakdown of glucose to lactate or ethanol (Cammack et al. 2008)
dilution
- the act or process of making more dilute (def. 2). (Cammack et al. 2008)
specific growth rate
- the steepness of a growth curve (sigmoidal), and it is defined as the rate of increase of biomass of a cell population per unit of biomass concentration. (Cambridge Dictionary. Accessed from: [1])
cis-regulatory motif
- a site that is bound by a 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. (Vazquez-Flota, F., & Loyola-Vargas, V. M. (2006). Plant Cell Culture Protocols.)

Outline of Paper

Main Results

Significance of the study

Shows the importance of separating phases in the physiological response to environmental change

Findings and limitations of previous studies

Previous studies showed that temperatures below optimal range slow down enzyme kinetics/cellular processes
- Also affect cell characteristics and processes (i.e. growth phase, respiration, lipid composition of membranes, trehalose content)
Time scale important to gauge response
- Sudden exposure to change leads to rapid highly dynamic stress response
- Prolonged exposure leads to acclimation
Previous low-temperature studies had many discrepancies, inconsistencies in expression
Trehalose accumulation only found in near-freezing temps (not all cold shock situations)
No specific transcriptional network has yet been identified
Previous studies used batch cultures, which don't allow for prolonged exposure to cold
- Chemostat cultures enable accurate control of the specific growth rate

The Experiment

Goal is to investigate steady-state acclimitized growth of the yeast at suboptimal temps
Data can be found at Genome Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under the series number GSE6190.

Strain and growth conditions

Prototrophic, haploid strain of S. cerevisiae
Grown at dilution rate of 0.03 h^-1 at both 12 or 30 deg C in 2.0 I chemostats
- Grown in a defined synthetic medium limited by carbon of nitrogen with all others in excess
- pH kept constant at 5.0
- Stirrer speed kept at 600 rpm
Constants: biomass dry weight, metabolites, dissolved oxygen, gas profiles

Analytical Methods

Culture supernatants obtained with the rapid sampling method outlined in Mashego et al. (2003)
Glucose and metabolite concentrations analyzed by high-performance liquid chromotography
- Also determined residual ammonium concentrations, ethanol evaporation, culture dry weights, whole cell protein contents
Measured trehalose and glycogen
- Triplicate trehalose measurements and duplicate glycogen for each chemostat

Microarray Analysis

Cells hybridized to microarrays
- Results for each growth condition derived from three independent replicates
  - Average coefficient of variation for each condition was below 0.20

Data Analysis

Pairwise comparisons
- Statistical analysis performed in Microsoft Excel running a significance analysis add-in for microarrays
- Created Venn diagrams and heat-maps of transcript data using Expressionist Analyst version 3.2
Promoter analysis performed using Regulatory Sequence Analysis Tools (web-based)
Created GO lists using Database for Annotation, Visualization, and Integrated Discovery 2006
Found overrepresentation of transcription factor binding sites using a Bonferroni correction and p-value threshold of 0.01

Figures and Tables

Table 1

Physiological characteristics of S. cerevisiae grown in ammonium and glucose limited anaerobic chemostat cultures
DNA microarray analysis used to analyze growth effects of temperature on gene expression in chemostat cultures
- Each culture grown at either 30 or 12 deg C and limited glucose or ammonium

Figure 1

Global transcriptome responses to anaerobic growth at 12 and 30 deg C in glucose and ammonium limited chemostat cultures
Venn diagram shows number of significantly differently expressed genes between 12 and 30 deg C in both conditions
- Out of 1065 temp-responsive genes, only 235 genes showed a consistent change in regulation in both conditions

Figure 2

Heat map representing the transcript level ratio of 1065 differentially expressed genes in anaerobic glucose and ammonium limited chemostat cultures grown at 12 and 30 deg C
- Categorizes genes by their regulation in all 4 conditions
Transport kinetics change was reflected in the transcript levels of genes under glucose limitation
- Increased transcript levels at 12 deg C

Table 2

Protein and storage carbohydrates contents of S. cerevisiae biomass
Same four conditions as before
- Measured contents of nitrogen, trehalose, glycogen, etc.
Trehalose and glycogen biosynthesis genes are induced by cold shock
- Once cells adapt to the temp, the up-regulation of these storage carbohydrates recedes

Table 3

Significantly overrepresented cis-regulatory binding motifs in 5' upstream regions and significantly overrepresented promoter elements that bind know TFs in low temp up and down regulated clusters from ammonium and glucose limited chemostat experiments
Separates by cluster (C-lim, M-lim, or both... Up or down reg)
(A) 5' upstream cis-regulatory motif
- Lists motifs, binding proteins, promoter elements, and occurrences
(B) Overrepresentation of transcription factors binding targets
- Lists factors, p-values, number of genes in this category in the cluster, and the same but in the whole genome
Clear enrichment of PAC cis-regulatory motifs
- Involved in transcriptional regulation of ribosomal protein encoding genes

Figure 3

Genes differentially expressed in batch cultures during adaptation to low temp
Venn diagram dhows the number of genes that are common to three batch-culture studies
Heat map shows transcript ratio of 259 common genes (small number)
- 91 genes commonly up-regulated
- 48 genes commonly down
- 120 genes differ

Figure 4

Comparison of the transcript ratio of 259 commons genes from above figure
Venn diagrams show number of low temp responsive genes common to the batch-culture and the chemostat-based datasets
Heat map shows expression ratios of the common genes
- Bracketed genes show consistent regulation in all datasets
Three genes encoding transporters found to be down-regulated
Five genes for protein translocation were up-regulated

Figure 5

Comparison of the genes specifically up or down regulated during acclimation/adaptation
Uses Venn diagrams to show common genes between groups
- Groups are batch studies, growth rate studies, this study
- Upper diagrams are down regulated
- Lower are up regulated
Used to discuss differences in results and discrepancies

Figure 6

Very similar to Figure 5
Many consistently up or down regulated genes in batch-culture found to match ESR genes identified by Gasch et al. (2000)
- However, in chemostat cultures, showed opposite response, suggesting alleviation of ESR at low temp
  - Shows that ESR is a response to sudden exposure to cold

Implications of the Study

Comparison to other studies

This study looked at acclimation of the yeast genes to consistent low temp, whereas other studies looked at adaptation to cold shock
- Revealed limited correspondences and important differences
  - Only clear similarity in gene groups was a group involved in lipid metabolism
  - Differences include changes in protein-folding, cold shock can cause incorrectly folded proteins, not as much a problem in low temp acclimatized yeast

Implications and further steps

This study demonstrated that transcriptional responses to low temp and low specific growth rate can be separated and looked at apart using chemostat cultures
- Shows importance of separating different phases of physiological adaptation to environmental change
In the future, could analyze post-transcriptional modes of cellular regulation using the same approach/similar methods

My Evaluation

Did a good job of supporting claims with literature citations and figures
- All the figures were clearly labeled and explained
They outlined their methods and the software they used well
- Could probably follow steps if I needed to

Acknowledgments

I would like to thank my partner for this assignment, Fatima. We texted a couple times about the assignment and talked about the figures together.

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

Alison S King (talk) 20:43, 27 March 2019 (PDT)

References

Biology Online Dictionary. (2005). Accessed from: ]https://www.biology-online.org/dictionary/Permease]

Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (2008) Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press.

Loyola Marymount University (27 March 2019) BIOL388/S19:Week 9. Retrieved from https://openwetware.org/wiki/BIOL388/S19:Week_9 on 27 March 2019.

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. [2].

Vazquez-Flota, F., & Loyola-Vargas, V. M. (2006). Plant Cell Culture Protocols.

Links

Alison S King

Class Page: BIOL388/S19

Assignment Pages:

Shared Journals:

Individual Journals: