Sahil Patel Week 9

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Electronic Lab Notebook

Purpose

The purpose of this assignment was to prepare for our second journal club which was on the article, Acclimation of Saccharomyces cerevisiae to Low Temperature.

Preparation for Journal Club 2

Key Terms

  • chemostats: The chemostat is an experimental apparatus where the chemical environment can be maintained static and nutrient availability can be controlled by the experimenter.
  • transcriptome: The transcriptome is defined as the complete set of transcripts in a cell, and their quantity, for a specific developmental stage or physiological condition
  • prototrophic strains: Strain's that have the same nutritional requirements as the wild-type strain.
  • cis-regulatory motif: A noncoding DNA sequence in or near a gene required for proper spatiotemporal expression of that gene, often containing binding sites for transcription factors. Often used interchangeably with enhancer.
  • diurnal: The condition of occurring or being active during the day.
  • mannoprotein: yeast cell wall components that are proteins with large numbers of mannose groups attached; highly antigenic.
  • supernatants: The soluble liquid fraction of a sample after centrifugation or precipitation of insoluble solids.
  • homeoviscous adaptation: Homeoviscous adaptation is a process that allows cell membranes to maintain a liquid-crystalline state at temperatures potentially low enough to cause a membrane to enter the gel state.
  • acclimation: adaptation to a new environment or to a change in the old.

Outline

Introduction

  • Studies have shown that temperatures below optimal range for growth slow down enzyme kinetics and thus cellular processes
  • Previous low-temperature studies also had inconsistent data and discrepancies in expression
  • In this study, chemostat cultures allow for an accurate control of the specific growth rate whereas before batch cultures were used which did not allow for a prolonged exposure to cold
  • The goal is to investigate steady-state, acclimatized growth of S. cerevisiae at suboptimal temperatures, with emphasis on genome-wide transcriptional regulation

Materials and Methods

Strain and Growth Conditions
  • The prototrophic, haploid reference S. cerevisiae strain CEN.PK113-7D was focus of study
    • grown at a dilution rate (D) of 0.03 h−1 at both 12 or 30°C in 2.0 l chemostats
  • Cultures were grown in a defined synthetic medium that was limited by carbon or by nitrogen with all other growth requirements in excess
  • Three individual replicates were taken per culture
Analytical Methods
  • Culture supernatants were obtained with the rapid sampling method
  • Concentrations of glucose and metabolites were analyzed by high-performance liquid chromatography on an AMINEX HPX-87H ion exchange column using 5 mM H2SO4 as the mobile phase
  • Glucose released by glycogen and trehalose breakdown was determined using the UV method
  • The elemental composition of the yeast biomass grown under nitrogen limitation was analyzed using the Carlo Erba elemental analyzer
Microarray Analysis
  • The results for each growth condition were derived from three independently cultured replicates
  • The average coefficient of variation for the triplicate transcriptome analyses for each of the four growth conditions was below 0.20
  • The level of the ACT1 transcript varied <12% over the four growth conditions
  • Overrepresentation of transcription-factor binding sites as defined by chromatin immunoprecipitation (ChIP)-on-chip analysis was assessed by Fisher's exact test, employing hypergeometric distribution with a Bonferroni correction and a p value threshold of 0.01
  • The microarray data have been deposited at Genome Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under the series number GSE6190

Results

Figures and Tables
  • Table 1
    • Physiological characteristics of S. cerevisiae grown in ammonium- and glucose-limited anaerobic chemostat cultures
    • Biomass yields and fermentation rates were similar at 12 and 30°C in both carbon- and nitrogen-limited chemostat cultures, indicating that growth efficiency was not severely affected by the growth temperature
  • Figure 1
    • Global transcriptome responses to anaerobic growth at 12 and 30°C in anaerobic glucose- and ammonium-limited chemostat cultures (D = 0.03 h−1)
    • The Venn diagram shows the number of significant differentially expressed genes between 12 and 30°C in both C and N limitations
  • Table 2
    • Protein and storage carbohydrates contents of S. cerevisiae biomass grown in ammonium- and glucose-limited anaerobic chemostat cultures
  • 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. Nineteen NCR-responsive genes are underlined
  • Table 3
    • Identification of (A) significantly overrepresented cis-regulatory binding motifs in 5′ upstream regions
    • (B) significantly overrepresented promoter elements that bind known transcription factors (TF) or TF pairs according to ChiP-on-chip analysis in low-temperature up- and down-regulated gene clusters derived from C-Lim and N-Lim chemostat experiments
  • Figure 3
    • Genes differentially expressed in batch cultures during adaptation to low temperature
    • (A) Venn diagram showing the number of genes that are common to three batch-culture studies on low-temperature transcriptional adaptation
    • (B) Heat map representing the transcript ratio of 259 genes found in common in the three batch-culture low-temperature transcriptome datasets
  • 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 h−1)
    • Venn diagrams show the number of low-temperature–responsive genes common to the batch-culture and chemostat-based datasets
    • The heat map shows the expression ratios of the genes common to batch and chemostat datasets
    • The genes indicated between brackets show a consistent transcriptional regulation at low temperature in all datasets
  • Figure 5
    • Comparison of the genes specifically up- or down-regulated during acclimation or adaptation to low temperature with the growth rate–dependent genes
  • Figure 6
    • Comparison of the genes specifically up- or down-regulated during acclimation or adaptation to low temperature with the ESR genes

Conclusion

  • This study looked at acclimation of the yeast genes to consistent low temp, whereas other studies looked at adaptation to cold shock
  • It demonstrated that transcriptional responses to low temp and low specific growth rate can be separated and looked at separately using chemostat cultures
  • Low-temperature acclimation in S. cerevisiae does not solely involve transcriptional reprogramming
  • For further exploration, the approach described in this study can also be used to analyze post-transcriptional modes of cellular regulation

Acknowledgments

  • My homework partner, Edward Talatala, and I communicated over text to discuss our figure and the research article for the journal club.
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

References

Sahil Patel (talk) 04:57, 28 March 2019 (PDT)

Other Assignments

Week 1: Instructions, Class Journal and User Page

Week 2: Instructions, Class Journal and Individual Assignment

Week 3: Instructions, Class Journal and Individual Assignment

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

Week 6: Instructions, Class Journal and Individual Assignment

Week 7: Instructions, Class Journal and Individual Assignment

Week 9: Instructions, Class Journal and Individual Assignment

Week 10: Instructions, Class Journal and Individual Assignment

Week 11: Instructions, Class Journal and Individual Assignment

Week 12: Instructions, Class Journal and Individual Assignment

Week 14/15: Instructions and Individual Assignment