Avalekander Week 9

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Purpose

The purpose of this journal club is to familiarize ourselves with research involving a chemostat-based transcriptome analysis in conjunction with the acclimation of yeast to cold stress. This article is meant to introduce us to the modelling of chemostat-based transcription in comparison to the batch-culture analysis described in the previous journal club.

Biological Terms

  1. Mannoproteins- yeast cell wall components that are proteins with large numbers of mannose groups attached; highly antigenic (https://www.biology-online.org/dictionary/Mannoproteins).
  2. Prototrophic strains- Strain's that have the same nutritional requirements as the wild-type strain (https://www.biology online.org/dictionary/Prototrophic_strains).
  3. Cryostat- A chamber that can maintain very low temperatures. Medical laboratories use a cryostat to preserve frozen tissue samples while a microtome, an extremely sharp cutting instrument mounted inside cryostats, slices the tissue into pieces thin enough to be observed under a microscope (https://www.medicinenet.com/script/main/art.asp?articlekey=12235).
  4. Applikon ADI 1030 Biocontroller- The Applikon Biotechnology ADI 1030 Bio Controller is a process controller, that is capable of measuring and controlling up to four parameters. The controller contains an 8 bit micro processor, loaded with flexible firmware that can control four parameters through eleven outputs. It features controller board, LCD display, keyboard, host computer, and etc (https://americanlaboratorytrading.com/lab-equipment-products/applikon-biotechnology-adi-1030-bio-controller_15939).
  5. Hypergeometric distribution- Hypergeometric distribution is the probability distribution of a hypergeometric random variable. Hypergeometric distribution can be defined as discrete probability distribution describing the probability of getting k successes in n draws without replacement where the sample population is N. Each draw is either success or failure and the population consists of exactly K successes. In statistics, the hypergeometric test uses the hypergeometric distribution to see the effect of k successes statistically (http://www.probabilityformula.org/hypergeometric-distribution.html).
  6. Chromatin immunoprecipitation- The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This assay can be used to identify multiple proteins associated with a specific region of the genome, or the opposite, to identify the many regions of the genome associated with a particular protein (https://www.cellsignal.com/contents/resources-applications-chromatin-immunoprecipitation/overview-of-chromatin-ip-assay-methodology/chip-assay-overview).
  7. cis-regulatory motifs- Cis-regulatory DNA sequences include two distinct elements: promoters/proximal elements and the distal regulatory regions including enhancers, silencers or repressors, insulators and locus control regions (LCRs). These elements act in co-operation with one another to govern a co-ordinated expression pattern of a gene (file:///C:/Users/circulation/Downloads/chapter1%20(1).pdf).
  8. Homeoviscous adaptation- Biological membranes are complex and dynamic assemblies of lipids and proteins. Poikilothermic organisms including bacteria, fungi, reptiles, and fish do not control their body temperature and must adapt their membrane lipid composition in order to maintain membrane fluidity in the cold. This adaptive response was termed homeoviscous adaptation and has been frequently studied with a specific focus on the acyl chain composition of membrane lipids (https://www.sciencedirect.com/science/article/pii/S0022283616303084).
  9. Diurnal- recurring every day; having a daily cycle; of, relating to, or occurring in the daytime (https://www.merriam-webster.com/dictionary/diurnal).
  10. Transcriptome- The initial product of genome expression is the transcriptome, a collection of RNA molecules derived from those protein-coding genes whose biological information is required by the cell at a particular time (https://www.ncbi.nlm.nih.gov/books/NBK21121/0).

Outline

Introduction

  • Tai et. al stated that despite the plethora of low-temperature transcriptome databases, major questions still need to be addressed. These questions included:
    • The major discrepancies between low-temp transcriptome data that has been published. These included inconsistency in the expression of ribosomal protein genes as one study showed increased transcription, however, in the Schade et al paper the temperature downshift caused the opposite to occur.
    • Trehalose is only necessary for survival at near freezing temps, however other genes involved in carbohydrate induction do not show this same trend.
    • The transcriptional low temp network for Msn2p/Msn4p complex has still not been identified.
    • The differences in transcriptional response to adaptation and acclimation to low temp have never been thoroughly investigated. Testing for prolonged exposure in batch experiments like in previous studies is not ideal.

Methods

  • Yeast were grown at a dilution rate of 0.03 h^-1 at both 12 degrees or 30 degrees celsius in 2.01 chemostats (incubator) with a working volume of 1.01. The cultures were grown in synthetic medium that was either limited by carbon or nitrogen with all other growth requirements available. The pH was kept constant at 5.0 and the stirrer was at 600 rpm.
  • The strain of S. cerevisiae was CEN.PK113-7D. The strain was haploid.
  • Yeast were grown at a dilution rate of 0.03 h^-1 at both 12 degrees or 30 degrees celsius in 2.01 chemostats with a working volume of 1.01. The cultures were grown in synthetic medium that was either limited by carbon or nitrogen with all other growth requirements available. The pH was kept constant at 5.0 and the stirrer was at 600 rpm.
    • 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 (NH4)2SO4, 5.75 g liter^-1 K2SO4, 3.0 g liter^-1 KH2PO4, 0.5 g liter^-1.
  • Time unfortunately was not specified.
  • The control was at 30 degrees C with ammonia and glucose.
  • Three independent steady-state chemostat cultivations were performed.
  • The methods Tai et al. used to prepare RNA, label, and hybridize it included sampling and probing cells, hybridizing it to Affymetrix Genechip microarray, determining RNA quality through the use of Agilent 2100 Bioanalyzer, and then analyzing the growth condition in triplicate.
Mathematical/Statistical Methods
  • SAM database was used for pair-wise comparison. Statistical significance was assessed using a threshold fold difference of 2 and a median false discovery rate of 1%. Expressionist Analyst generated Venn diagrams and heat map visualization of data. The DAVID database was used to assess the overrepresentation of GO biological processes categories.
  • Fisher's test was used to assess the overrepresentation of transcription-binding sites by the (chIP)-on-chip analysis. The Fisher test used hypergeometric distribution with a Bonferroni correction and a p-value threshold of 0.01.
  • The microarray data is publically available at Expression Omnibus database under series number GSE6190.

Results

  1. Table 1: This table displays the limiting nutrient on the y-axis and the physiological data obtained for the various tests. Measurements were made by calculating the mean =/- the standard deviation for the three trials. Reduced growth does not appear to be affected by temperature.
  2. Figure 1: This figure does not necessarily have axes but it depicts a venn diagram showing the numbers of significantly different genes down and up-regulated under Nitrogen deficiency and or glucose (carbon) deficient conditions during the cold shock. The measurements were made through analysis of DNA microarray data. This figure shows that the temp response is strongly context dependent with respect to the nutrient limitation regime.
  3. Figure 2: This figure identifying the regulatory networks involved in the acclimation of yeast to low temps was generated by screening the temp responsive genes for enrichment of specific functional categories and their promotor regions were searched for cis-regulatory motifs.This figure expands on figure 1 by looking further into which genes specifically were up/down-regulated at the different temps and nutrient levels, as well as what these genes regulate such as lipid metabolism, etc. Change in transport kinetics was reflected in the transcript levels of genes involved in the uptake of growth limiting agents. This was showed as the hexose-transporting genes under glucose deficiency were increased transcprition compared to at 30 degrees in which transcription decreased.
  4. Table 2: This table depicts the protein and storage contents in the yeast biomasss grown in ammonium and glucose-limited anaerobic chemostat cultures. The different limiting nutrients are shown with the mean +/- standard deviation of the data from the 3 chemostat cultivations.
  5. Table 3: Part A represents the overrepresented cis-regulatory binding motifs in the 5' upstream regions. The regulatory cluster along with the motif name, promoter and binding protein are also displayed along with the number of occurrences of the promoter element in a cluster, compared with the expected value and the probability of finding that same number of patterns by chance alone. Part B showed the overrepresented promoter elements that bind transcription factors according to chiP-on-chip analysis in the low temp up/down-regulated gene clusters from the nutrient deficiency experiment. At 12 degrees C among the genes that showed increased transcription at the 5' upstream sequence in up regulation of PAC cis-regulatory motifs.
  6. Figure 3: Part A shows a Venn diagram of the genes that are common to all three batch-culture studies on low-temp transcriptional adaptation. Part B shows a heat map of expression ratios of the 259 genes that the studies had in common. Gene regulation was then examined based on analysis of the microarray data and the commonly up-regulated, commonly down-regulated, as well as the 120 genes that were differentially regulated are named.
  7. Figure 4: This figure shows the comparison of the transcript ratio of 259 genes that the chemostat experiment in this study had in common with the batch experiments. It displays this in the form of Venn diagrams and then a side-by-side comparison of the microarray expression ratios. In brackets, it shows the consistent transcriptional regulatory factors that appear to regulate cold response for all data sets.
  8. Figure 5: These Venn diagrams depict the genes commonly up/down-regulated during adaptation from previous studies compared to the gene regulation during acclimation which was studied in this research paper. There was insignificant overlap between the comparison Venn diagrams as the numbers were not close.
  9. Figure 6: This Venn diagram depicts the genes found in common in previous studies that up/down regulate and compares those to the ones found in this study of acclimation. The comparison shows a strong correlation between the two sets.

Conclusions

  • The chemostat study compares data with the following other publications: Schade et al. (2004), Murata et al. (2006), Sahara et al. (2002), and Gasch et al. (2000). The number of significantly changed genes found by Sahara was 1609 genes, 2339 genes for Murata, and Schade used only provided ESR genes from transcripts.
  • Chemostat studies are more accurate compared with batch cultures because in batches the exposure to low temps induces increased storage carbohydrates, however in the chemostat study storage of carbohydrates involves the Msn2/Msn4 complex. Low temp acclimatized growth does not involve this same complex.
  • Transcription of ESR genes is elicited by a reduced specific growth rate, rather than by low temps.
  • The 235 genes showed a similar transcriptional response to cold temp despite differences in the growth limiting nutrient.
  • Lipid metabolism was the only common function of the group of genes that consistently regulated transcription in cold temps for both batch and chemostat studies.
  • Significant differences between transcriptional response at low temps occur between long term, adapted exposure in comparison to abrupt temperature change.
  • Chemostat studies can take two intrinsically related parameters and separate them, whereas batch-cultures cannot do this.
  • Discriminating different phases of physiological adaptation is important.
  • I feel that the authors should maybe do this same type of experiment with heat shock and the sudden effect compared to after acclimation and see if those results are consistent with the results of this study.
Critical Evaluation
  • I did think that this paper was better at backing up their claims and findings than the Schade paper and thought that overall the data was well supported. However, in the materials and methods section, I wish that they had discussed the methods more in depth rather than just linking to other articles that explained the same method. Although this would have made the paper longer, sometimes I was a bit confused by their techniques and looking at all of the previous articles to understand seems like a lot to expect of the reader. Also, the amount of time yeast were exposed to cold was not included and this is a really key aspect of the study so I am unsure why this was not included.

Acknowledgements

I would like to acknowledge my homework partner, Austin. I would also like to acknowledge my professors, Dr. Dahlquist and Dr. Fitzpatrick for their expertise and assistance.

Except for what is noted above, this individual journal entry was completed by me and not copied from another source. Avalekander (talk) 20:18, 27 March 2019 (PDT)

References

Biology 388 Assignments

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