Austindias Week 9

Purpose

The purpose of this assignment is to dissect and comprehend this research paper and connect the ideas presented to previous research we have studied. We will use this information to participate in an intellectual conversation about the research project as a whole.

Definitions

1. Mannoproteins: are polysaccharides released from yeast cells during fermentation and by autolysis (Robinson and Harding, 2015).
2. Batch Culture: A technique used to grow microorganisms or cells. A limited supply of nutrients for growth is provided; when these are used up, or some other factor becomes limiting, the culture declines. Cells, or products that the organisms have made, can then be harvested from the culture (Martin and Hine, 2015).
3. Chemostat: An apparatus allowing the continuous cultivation of bacterial populations in a constant, competitive environment. Bacteria compete for a limiting nutrient in the medium (King et al., 2013).
4. Prototroph: any strain of a microorganism (alga, bacterium, or fungus) that does not require any substances in its nutrition additional to those required by the wild type (Cammack et al., 2006).
5. Transciptome: The full complement of RNA transcripts of the genes of a cell or organism (Martin and Hine, 2015).
6. Permeases: A general term for a membrane protein that selectively increases the permeability of the plasma membrane to a particular molecule (Lackie, 2010).
7. Motif: A small structural element or domain that is recognizable in several proteins (Lackie, 2010).
8. Monod Kinetics: An unstructured model used to describe the correlation of substrate concentration with microbial growth kinetics. The model is based on enzymatic Michaelis–Menten kinetics (Schaschke, 2014).
9. Supernatant: The fluid lying above a precipitate in a centrifuge, following the centrifugation of a suspension (King et al., 2013).
10. Immunoprecipitation: The precipitation of a multivalent soluble antigen by a bivalent antibody, resulting in the formation of a large complex which precipitates when the ratios of antibody and antigen are near equivalence (Lackie, 2010).

Outline

Background

• S. cerevisiae commonly resides in sugar rich environments such as plant environments
• Low temperatures affect cell growth phases (Werner-Washburne et al., 1993), respiration (Lewis et al., 1993), and membrane composition.
• Extended exposure to nonlethal stimuli leads to acclimation by affected cells such as adaptation of genome expression.
• Two phases of cold response, early and late.
• Genes often associated with cold shock and heat shock
  • HSP12, HSP26, HSP42, HSP104, YRO2, and SSE2
• Many genes that are regulated during other environmental stresses are often regulated in cold shock.

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

• Discrepancies in previous cold shock experiment results
  • Ribosomoal protein genes
  • trehalose accumulation
  • Msn2p/Msn4p complex
• Differences in transcriptional response to adaptation and acclimation to cold shock has not been studied adequately
• Batch cultures used in previous studies is not effective for use in studying long term effect of cold shock because it does not account for the effect of temperature on transcription
• Use of chemostat cultures in this experiment allows control of growth rate as well as allowing highly reproducible data

Methods

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

• Yeast cells grown at a dilution rate (D) of 0.03 h1at 12 C or 30 C in 2.0 l chemostats
• Cultures were grown in a defined synthetic medium (described below) that was limited by carbon or by nitrogen with all other growth requirement in excess.
• pH remained at 5.0 by automatic addition of 2 M KOH
• stirrer included at 600 rpm
• Anaerobic conditions

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

• haploid S. cerevisiae strain CEN.PK113-7D

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

• Bacteria grown in a defined synthetic medium that was limited by carbon or by nitrogen with all other necessary components in excess according to (Tai et al., 2005).
  • carbon-limited cultivation= 5.0 g liter^-1(NH4)2SO4, 3.0 g liter-1 KH2PO4, 0.5 g liter^-1 MgSO47H2O, and 25 g liter^-1 glucose
  • nitrogen-limited cultivation= 0.65 g liter^-1 glucose, 5.75 g liter^-1 K2SO4, 3.0 g liter^-1 KH2PO4, 0.5 g liter^-1
• grown at 12 C in chemostat device under anaerobic and steady-state conditions
• they do not specify the length of time bacteria were exposed to temperature change

What controls did they use?

• 30 C with ammonia and with glucose

How many replicates did they perform per treatment or timepoint?

They performed three replicates for each growth condition

What method did they use to prepare the RNA, label it, and hybridize it to the microarray? What mathematical/statistical method did they use to analyze the data?

• Average coefficient of variation
• Microsoft excel with micro-array significance add in, SAM version 1.12
• Created Venn-diagrams and heat maps using Expressionist Analyst version 3.2
• Promoter analysis done using web-based software Regulatory Sequence Analysis (RSA) Tools

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

• The data is available publicly for download at http://www.ncbi.nlm.nih.gov/geo/ under series number GSE6190

Results

Table 1

This table depicts the physiological characteristics of S. cerevisiae grown in -ammonium and glucose-limited anaerobic chemostat cultures at 12 C and 30 C. The values reflect the average of the three replicates +/- the standard deviation. The standard deviations are relatively small because these characteristics were held constant. Biomass yields denoted by Y Glu/X, along with fermentation rates were similar at 12 C and 30 C in both -carbon and -nitrogen cultures suggesting that growth was not heavily impacted by growth temperature.

Figure 1

This figure illustrates a Venn-diagram expressing transcriptome responses to anaerobic growth at 12 C and 30 C in anaerobic -glucose and low ammonium cultures. In total there were 1065 genes that showed significant expression in response to temperature. 235 genes out of 1065 were found to be in common between the carbon limited and nitrogen limited culture. Of these similar genes, 96 were up regulated and 139 were down regulated. 571 genes were found to show significant expression in only carbon limitation. Whereas, 259 genes displayed significant expression in ammonia limitation exclusively. A large number of genes did not show significant change in response to temperature change. This figure shows that there is a clear difference in the genes involved in -carbon and -ammonium treatments when exposed to temperature change, considering only 235 genes were consistent between both groups.

Figure 2

This figure illustrates gene expression behavior under different conditions. The x axis contains the type of treatment such as nitrogen limitation or carbon limitation at corresponding temperatures of 12 C and 30 C. The heat map on the y axis shows whether the specific group of genes was induced, repressed, or had no change from the initial time point. In the figure genes are clustered by their behavior in these conditions. In addition, go terms are included to denote the functions of the genes of each cluster. Figure 2 acts as a heat map visualization of the information presented in Figure 1. NCR responsive genes are underlined. NCR stands for nitrogen responsive genes, which allows repression of genes involved in the utilization of poor nitrogen sources when preferred ones are available. A few conclusions can be drawn from this figure:

• Increased concentration of limiting nutrients at 12 C resulted in higher catabolite repression indicated by down regulation of common genes for glucose repression and nitrogen repression
• Genes related to ribosome biogenesis and assembly showed higher induction at 12 C than at 30 C in both -glucose and -ammonium cultures
• Response was most striking in the ammonium-limited cultures, where an additional 80 genes involved in protein synthesis increased expression at 12 C
• Increase of the rRNA content at low temperature in the ammonium-limited cultures could be due to increase transcript levels of four genes that encode subunits of polymerase I
• Genes involved in membrane transport processes were strongly overrepresented among the genes that were repressed at low temperature

Table 2

Table 2 regards S. cerevisiae biomass components of protein and storage carbohydrates grown under -ammonium and -glucose anaerobic conditions. The dependent variables include the averages of the following: temperature, biomass dry weight, amount of protein in the cell, nitrogen content, trehalose content, and glycogen levels. Again standard deviation is included, but since these conditions are kept at steady state, these values are small. The limiting nutrients and corresponding temperatures are included as the independent variables. Table 2 shows that in the -ammonium cultures, trehalose and glycogen levels were significantly lower at 12 C in comparison to 30 C. While, cellular proteins and nitrogen content were significantly higher at 12 C than 30 C.

Table 3

This table focuses in on regulatory binding motifs in 5' upstream regions and over representation of transcription factors binding targets. For both topics the same gene clusters from Figure 2 are used to separate the genome. For regulatory binding motifs researchers looked at the promoter element, number of occurrences of promoter element in the respective cluster, the expected number of occurrences of the promoter element in a randomly chosen cluster of genes of the same cluster size, and the probability of finding the number of patterns with the same degree of over representation, based on chance alone. For over representation of transcription factor binding targets the research team considered the p values of each TF, number of genes in this category within the specific cluster, and the number of genes in this category within the entire genome. Table 3 leads to the following three conclusions:

• down regulation of the targets of NCR could be supported by the enhancement of GLN3 and GLN3/DA182 targets and over representation of the protein binding motifs "AAGATAAG". This is comparable to the "GATAAG" binding motif related to the NCR method
• over representation of STRE elements within upstream areas of genes reveals lower expression level at 12 C in -ammonia cultures
• of the genes that did show increased transcript level at 12°C, there was an enrichment of PAC cis-regulatory motifs

Figure 3

Figure 3 looks to compare the results at low temperature of studies that used batch cultures. 91 genes were discovered to be commonly up regulated. 48 genes were found to be commonly down regulated. 120 genes were found to be deferentially regulated between the three batch experiments. For each type of similarity, they include a full list of all the genes that were found to be in common. This figure shows that the three data sets found 259 genes in common, but these genes responded differently at times. This figure is helpful to suggest why chemostat cultures are better than batch cultures, since results are not affected by various environmental stimuli that may lead to inconsistency in gene behavior that is not necessarily related to temperature alone.

Figure 4

Figure 4 compares the chemostat culture expressions at low temperatures to all three batch culture results. On the x axis the various time points used in each experiment are included and on the y axis a heat map helps visualize the gene expression behavior. The heat map on the left shows genes that were up regulated at low temperatures in this experiment and the heat map on the right displays genes that were down regulated at low temperatures in -glucose and -ammonia conditions. They then compared the results for these specific genes to the results of each batch culture experiment. A Venn diagram is also utilized to help display similarities and differences between batch experiments and this chemostat experiment. The main conclusions that can be drawn from Figure 4 are:

• only 11 genes showed a consistent pattern of regulation between all experiments
  • up regulated: PIR3, SFK1, YPC1, YEL073C, YNL024C, and YLR225C whereas
  • down regulated: PHO84, FUI1, AHA1, FCY2, and YLR413W
• it is difficult to replicate data using different methods of experimentation

Figure 5

Figure 5 consists of Venn-diagrams that compare and contrast growth-dependent genes between the chemostat and batch studies with studies by Castrillo et al. (2007) and Regenberg et al. (2006). The authors denote batch studies as adaptation and the chemostat study as acclimation. For both comparison groups there is a very small number of genes that overlap between growth rate studied and the respective batch and chemostat studies. However, there were more genes in common between the growth-dependent studies and the batch studies, than the chemostat study. The comparison conducted between temperature-responsive genes and genes within the chemostat study showed negligible overlap at 0.7%.

Figure 6

Figure 6A has two circles for results from batch data. One of these circles states the number of genes that were up regulated, while the other consists of the genes that were down regulated. The Venn diagram on the left of Figure 6A includes ESR data for genes that were up regulated in response to environmental stress. The intersection between circles represents the number of genes that were found to be in common. When comparing batch studies to genes found to be up regulated in the ESR study by Gasch, there were no genes that were found to be in common with the up or down regulated genes in the batch studies. The same result was found when comparing up or down regulated batch genes to down regulated ESR study genes. Figure 6B follows the same format as 6A, except instead of using batch gene data, it uses the genes found to be up or down regulated in this study. Similarly to batch comparisons, there were no genes found to be consistent among the comparisons. Even though there was no overlap between all three criteria in these instances, overlap did occur between two criteria in many cases. In addition, there was discrepancy between different studies regarding gene expression. Some of these relationships are summarized below:

• 50 % of the low-temperature up-regulated genes and 13% of the low-temperature down regulated genes found in the batch studies were also found in the ESR genes identified by Gasch et al (2000).
• 1/3 of low temperature genes found in the batch studies are connected to ESR genes
• 233 genes were up or down regulated in the study by Gasch et al. (2000) showed an opposite transcriptional response in the low-temperature chemostat

cultures

The main conclusion that can be drawn from this figure is that ESR is not a required response to growth at low temperature, but happens during adaptation to abrupt exposure to temperature change.

Discussion

Main Findings

• It is not possible to only change a single parameter without an effect on any others, even using the chemostat culture approach
• The only indistinguishable group of genes that was similarly regulated in low-temperature chemostats and batch culture studies on low-temperature adaptation were related to lipid metabolism
• Trehalose is not involved in steady-state low-temperature adaptation
• Transcript levels of environmental stress response genes were reduced at 12 C
• Significant differences between transcriptional response to low temperature occur between long term exposure in comparison to abrupt temperature change
• Transcriptional responses to low temperature and low specific growth rate can be separated by using chemostat cultures instead of batch cultures
• It is highly important to differentiate phases of physiological adaptation in response to environmental change
• Response to low temperatures by S. cerevisiae is not entirely dependent on changes in transcription

Importance of Study

This study implies that chemostat cultures may be more effective at studying yeast response at low temperature, rather than batch cultures used in previous studies. This study is important because it provides an alternative method for testing low temperature and its effect on gene expression, while keeping other variables relatively constant. In previous studies, growth rate may have had an affect on transcription behavior, rather than just temperature alone. This experiment is a step in the right direction as far as zoning on on the genes that are solely responsible for the cold shock response.

Critical Evaluation

I believe the authors of this research were successful in relaying their message of why chemostat cultures may be superior to batch cultures in studying the effect of temperature on gene transcription. However, a considerable red flag was apparent within the presentation of this research. The concern involves the amount of time bacteria were exposed to low temperature. The researchers use comparisons to other data collections very often in this paper, but as an audience we are unsure if this data is comparable because it may reflect completely different units of time. Even though they present differences in up or down regulation, we are unsure at what times these behaviors are occurring. It would be expected that expression would change at different time points during cold shock and recovery. I believe the inclusion of time points in their paper would have helped create clarity and solidified their reasoning of why a chemostat culture is an improvement over batch cultures. In the future, the authors should consider testing other parameters besides -ammonia and -glucose. In addition, I think it would be interesting to consider in allowing for aerobic respiration to occur to evaluate if there is a difference between how yeast respond to low temperatures in oxygenic conditions versus an-oxygenic conditions.

Acknowledgments

• I would like to thank my homework partner who Ava Lekander for helping clarify ideas within the paper over text message. I would also like to thank Dr.Fitzpatrick and Dr. Dahlquist for providing the research paper to be discussed and the necessary background knowledge to comprehend its content.

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

Austindias (talk) 17:08, 27 March 2019 (PDT)

References

Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (2006). Oxford Dictionary of Biochemistry and Molecular biology (2nd ed.). Oxford: Oxford University Press.

Dahlquist, K. and Fitzpatrick, B. (2019). BIOL388/S19:Week 9. [online] openwetware.org. Available at:Week 9 Assignment Page [Accessed 26 March 2019].

King, R. C., Stansfield, W. D., & Mulligan, P. K. (2013). A dictionary of genetics. Oxford University Press, USA.

Lackie, J. (2010). A dictionary of biomedicine. Oxford University Press.

Martin, E., & Hine, R. (Eds.). (2015). A dictionary of biology. Oxford Quick Reference.

Robinson, J., & Harding, J. (Eds.). (2015). The Oxford companion to wine. American Chemical Society.

Schaschke, C. (2014). A dictionary of chemical engineering. OUP Oxford.

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.

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