Falghane Week 9

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Purpose

The purpose of this assignment is to learn about the chemostat based transcription. This paper is a study involving chemostat and how it influences genes in both nitrogen and carbon-limited media.

Prepration for Journal Club 2

Biological Terms

  1. Genome: The complete set of genetic material in an organism (2016).
  2. Dilution: In microbiologic techniques, a method for counting the number of viable cells in a suspension; a sample is diluted to the point where an aliquot, when plated, yields a countable number of separate colonies (2015).
  3. Trehalose: A disaccharide made up of two α-glucose molecules, and serves as an energy source in certain fungi, bacteria, plants, and invertebrates (2019).
  4. steady state: A dynamic equilibrium (2005).
  5. Acclimation: adaptation to a new environment or to a change in the old. Adaptation to a new climate (2005).
  6. Transcription: The process of transcribing or making a copy of genetic information stored in a DNA strand into a complementary strand of RNA (messenger RNA or mRNA) with the aid of RNA polymerases
  7. Adaptation: The adjustment or changes in behavior, physiology, and structure of an organism to become more suited to an environment; the state reached by the biological population undergoing adjustments or changes (2018).
  8. Membrane Transport Protein: A type of protein that actively transports materials across a plasma membrane that would not otherwise allow this to occur(2012).
  9. Protein Synthesis: Protein synthesis is a process of creating protein molecules. In biological systems, it involves amino acid synthesis, transcription, and translation (2017)
  10. Fermentation: An anaerobic (without oxygen) cellular process in which organic food is converted into simpler compounds, and chemical energy (ATP) is produced (2017).

Outline of The Article

  • The importance or significance of this work is: To investigate steady state acclimatized growth of S. cerevisiae at suboptimal temperatures with emphasis on genome wide transcriptional regulations.
  • The limitations in previous studies that led them to perform this work were:
    • Inconsistency in the of expression ribosomal protein (RP) genes. Also although the induction of genes involved in reserve carbohydrate seems to be a consistent feature of cold shock, trehalas accumulation is only indispensable for survival in near-freezing conditions, above 10 C, a tps1 change and tps2 change double null mutant showed no growth defects or viability loss. Additionally, no clear low temperature specific transcriptional network has been identified thus far and the difference between the transcriptional response to adaptation and acclimation to low temperature was never thoroughly investigated before.
  • Treatment of the Cell: growing them in mediums limited by carbon or nitrogen.
  • Strain of yeast used: Haploid, S.Cerevisiae
  • The media they grow them in, temperature, type of incubator, and for how long:
    • S. cerevisiae was grown in anaerobic in 2.01 chemostat cultures at a fixed dilution rate of 0.03h-1
    • Ph was constant at 5.0 and KOH was automatically added at both 12 and 30 °C chemostats.
    • Controlled cultures grown at 12 c were grown in a medium limited by carbon and nitrogen with all other requirements in excess.
    • Stirrer speed was 600 rpm.
  • replicates performed per treatment or timepoint:
    • Four growth conditions were performed, and three independent cultured replicates were done for each growth condition.
  • method used to prepare the RNA, label it and hybridize it to the microarray:
    • The method they used to prepare the RNA, label it and hybridize it to the microarray is the same one that was used by Piper et al. And they determined the RNA quality using Agilent 2100 Bioanalyzer.
  • Mathematical/statistical methods used to analyze the data:
    • Microsoft Excel was used to run the significance analysis of microarrays add-in.
    • Promoter analysis was performed using a web-based software regulatory sequence Analysis.
    • Statistical assessment was done using the Database for Annotation, Visualization and Integrated Discovery (DAVID).
    • Overrepresentation of transcription-factor binding sites was assessed by Fisher’s exact test with a Bonferroni correction and a p-value threshold of 0.01.

The important implications of this work:

  • Consistent transcriptional response to low temperature, irrespective of the growth-limiting nutrients.
  • Transcriptional responses to low temperature and low specific growth rate, two parameters that are intrinsically linked in batch cultures can be dissected using chemostat cultures.
  • Low-temperature acclimation in S. cerevisiae does not solely involve transcriptional reprogramming.

Critical evaluation of how well I think the authors supported their conclusions with the data they showed. Are there any major flaws to the paper?

  • I think the conclusion was well supported
  • In the method section and specifically the method used to prepare the RNA, they chose to say that the method used was the same as the one that “Piper et al”. Used. They did so without explaining what the method was which I found confusing because it would mean that the reader must also read “Piper et al” paper and specifically their methods section in order to know what the method was which I think is not efficient.

This Work Compared with Previous Studies:

  • the sets of genes that were consistently up or down regulated at low temperature in batch cultures were like the ESR genes defined by Gesh et Al. However, the genes that showed a different transcript level at low temperature found by this study were different from the ones defined by Gesh et Al.

Future Directions the Author should Take:

  • In the future the author should explore the genes that showed a different transcript level at low temperature and redo that experiment in order to see if the genes resulting would still be different from the one defined by Gesh et Al. Also they should explore the confounding variables and see if they could be the reason behind the different results than Gesh et Al’s results.

Result that was Shown in each of the Figures and Tables:

Table1: the Physiological characteristics of S.Cerevisiae grown in ammonium and glucose limited anaerobic chemostat cultures.

  • What X and Y represented:
    • The table shows limiting nutrients glucose and ammonium at temperature 12 °C and 30 °C and the values represent the mean +/- standard deviation of data from three independent steady-state chemostat cultivation.
  • How the measurements were made:
    • By growing chemostat cultures at a fixed dilution rate of 0.03 h-1 and then measuring biomass yields and fermentation rates.
  • Trends shown by the plots and conclusions that can you draw from the data:
    • Biomass yields and fermentation rates were similar at 12 and 30 C in both carbon and nitrogen chemostat cultures which indicates that the efficiency of growth was not much affected by growth in temperature.


Figure 1: Global transcriptome responses to anaerobic growth at 12 and 30 °C in anaerobic glucose and ammonium limited chemostat cultures.

  • What X and Y represented:
    • The figure shows the number of significant differentially expressed genes between 12 and 30 °C in both carbon and nitrogen limitation.
  • How the measurements were made:
    • 494 genes in glucose-limited cultures and 806 genes in nitrogen-limited cultures
  • Trends shown by the plots and what conclusions can you draw from the data:
    • Strong context dependency with respect to the nutrient limitation region


Figure 2: Heat map representing the transcript level ratio of 1065 differentially expressed genes in anaerobic glucose- and ammonium-limited chemostat cultures (D = 0.03 h−1) grown at 12 and 30°C. The genes indicated in the figure belong to the enriched GO categories

  • What X and Y represented:
    • Y-axis represent the anaerobic carbon/ nitrogen-limited chemostat cultures at 12 and 30 C.
    • X-axis represents ration of 1065 differentially expressed genes.
  • How the measurements were made:
    • the temperature-responsive genes were screened for the enrichment of specific functional categories, and their promoter regions were searched for cis-regulatory motifs.
  • Trends shown by the plots and what conclusions can you draw from the data:
    • Change in transport kinetics can be reflected in the transcript levels of genes involved in the uptake of the growth-limiting nutrients
    • The adaptation was most striking under glucose limitation.


Table 2: showing protein and storage carbohydrates contents of S.erevisiae biomass grown in ammonium and glucose limited anaerobic chemostat cultures.

  • What X and Y represented:
    • Y-axis shows limiting nutrient
    • X-axis shows growth temp, biomass dry weight, whole cell protein, biomass nitrogen content, trehalose, and glycogen.
  • How the measurements were made:
    • Genes involved in the metabolism of storage carbohydrates and especially trehalose were observed after the cold shock and exposed to near freezing conditions.
  • Trends shown by the plots and what conclusions can you draw from the data:
    • trehalose and glycogen contents were significantly lower at 12 than at 30 c in ammonium limited chemostat cultures. And so were the trehalose contents lower at 12 °C in glucose-limited cultures.
    • Glycogen content was 50% higher in glucose-limited cultures grown at 30C.
    • Conclude: accumulation of glycogen and trehalose and transcriptional induction of genes that are involved in the synthesis of these compounds is not a prerequisite for yeast acclimation to low temperature.


Table 3: is the identification of (A) significantly overrepresented cis-regulatory binding motifs in 5’ upstream regions and (B) significantly overrepresented promoter elements that bind known transcription factors (TF) pair.

  • What X and Y represented:
    • Y-axis in table (A): regulatory cluster
    • X-axis in table (A): motif name, putative binding protein, promoter element, occa , expected occa , occ Ec
    • Y-axis in table (B): regulatory cluster
    • X-axis: factor, p-value, Kd, Fe
  • How the measurements were made:
    • Chemostat cultures were grown at different temperatures and genes were observed.
  • Trends shown by the plots and what conclusions can you draw from the data:
    • Among genes that showed an increase in transcription levels at 12 °C, they showed a clear enrichment of PAC cis-regulatory motifs which is involved in the regulation of transcription of ribosomal protein-encoding genes.


Figure 3:

  • What X and Y represented:
    • Y-axis: different genes.
    • X-axis the expression of the genes at the three different cultures
  • How the measurements were made:
    • Measuring genes response to downshift.
  • Trends shown by the plots and what conclusions can you draw from the data:
    • Few genes were constantly down/up-regulated at low temperature.
    • Lowering the temperature negatively impacts the function of the genes.


Figure 4: Comparison of the transcript ratio of 259 genes common to three batch-culture low-temperature transcriptome datasets with the 12°C/30°C ratio of the genes specifically up- (n 96) and down-regulated (n 139) in anaerobic glucose- and ammonium-limited chemostat cultures (D 0.03 h1).

  • What X and Y represented:
    • X-axis: results from different studies
    • Y-axis: genes that are commonly upregulated and downregulated in C and N limited media at 12 and 30 ° C. Genes in brackets show consistent transcriptional regulation at low temperature in all datasets.
  • How the measurements were made:
    • By comparing the ratio of 259 genes common to three batch culture low-temperature transcriptome datasets with 12° C/30° C ratio of genes specifically up- (n 96) and down-regulated (n 139) in anaerobic glucose- and ammonium-limited chemostat cultures.
  • Trends shown by the plots and what conclusions can you draw from the data:
    • Three genes that encode for transporters were found among the five consistently down-regulated genes.
    • Genes that were commonly regulated in a low-temperature chemostat were involved in lipid metabolism.


Figure 5: comparison of genes specifically up or down regulated during acclimation

  • What X and Y represented:
    • X-axis: adaptation and acclimation
    • Y-axis: comparison of the genes
  • How the measurements were made:
    • Quantify the genes that were down-regulated and up-regulated at 12 C.
  • Compare to previous studies.
    • trends are shown by the plots and what conclusions can you draw from the data:
    • The growth rate was not influenced by temperature but by glucose limitation.


Figure 6:

  • What X and Y represented:
    • Y-axis: acclimation and adaptation
    • X-axis: results of genes being up regulated or down regulated during acclimation or adaptation with the ESR genes from different studies
  • How the measurements were made:
    • Quantify the genes that were down-regulated and up-regulated at 12 C.
  • Trends shown by the plots and what conclusions can you draw from the data:
    • a third of the low-temperature-responsive genes found in the three batch-culture studies could be linked to ESR

Acknowledgments

  • I worked with my homework partner Alison S. King to better understand the project.
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Falghane (talk) 21:22, 27 March 2019 (PDT)

Refrences

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