Lkelly9 Week 3

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Electronic Lab Notebook

Purpose

The purpose of this assignment is to build our journal club skills by defining terms that we are not familiar with, outlining the paper, and reflecting on the experience in preparation for discussing the article with other classmates during lecture.

Preparation for Journal Club 1

Definitions

  • Continuous Culture: Also referred to as a batch culture; A large-scale closed system culture in which cells are grown in a fixed volume of nutrient culture medium under specific environmental conditions up to a certain density in a tank or airlift fermentor, harvested and processed as a batch, especially before all nutrients are used up.
  • Glutamine: A crystalline amino acid occurring in proteins; important in protein metabolism. One of the 20 amino acids that are commonly found in proteins. It is the amide at the carboxyl of the amino acid glutamate. It can participate in covalent cross linking reactions between proteins by forming peptide like bonds by a transamidation reaction with lysine residues.
  • Proline:One of the 20 amino acids directly coded for in proteins. Structure differs from all the others, in that its side chain is bonded to the nitrogen of the amino group, as well as the carbon. This makes the amino group a secondary amine and so proline is described as an imino acid. Has strong influence on secondary structure of proteins and is much more abundant in collagens than in other proteins, occurring especially in the sequence glycine proline hydroxyproline. A proline rich region seems to characterize the binding site of SH3 domains

Outline

  • Main result presented in the paper
    • The major finding of this paper was that nitrogen metabolism is regulated by the concentration of ammonia on various levels (physiology, transcription, and enzymes)
    • This paper ruled out the possibility of ammonia flux regulating nitrogen metabolism
    • Points to regulation by intra/extracellular concentrations of ammonia or changes in alpha-ketoglutarate, glutamate, or glutamine
  • Importance/Significance of this work
    • Understanding the effect of ammonia concentration on the nitrogen metabolism of S. cerevisiae can positively influence the food industry, as this type of yeast is used to make bread, beer, and other products.
    • Increasing the ammonia concentration in the right way can maximize biomass of S. cerevisiae.
  • Limitations in previous studies that led them to perform this work
    • Previous studies using continuous cultures have concluded that ammonia concentration is the main influence of nitrogen metabolism, but these cultures did not account for differences in external ammonia concentration or the rate or ammonia assimilation (ammonia flux). This indicates that it is possible that the flux, rather than concentration, is the main factor for nitrogen metabolism.
  • Methods used in the study
    • Physiological parameters
      • S. cerevisiae was grown in a continuous culture (as described the the above definition). The feeds had different concentrations of ammonia, ranging from 29-118 mM, a constant glucose concentration (100 mM), and the dilution rate was 0.15 h-1.
      • As the ammonia concentrations were fluctuated, the biomass concentrations were measured in order to observe the effect of ammonia concentration on biomass.
        • Biomass increase was observed when the concentration of ammonia was increased form 29 to 61 mM (ammonia was the limiting factor), but the biomass remained constant and glucose became limiting when the ammonia concentration in the feed exceeded 61 mM.
      • The ammonia flux was calculated using the biomass, ammonia concentration in the feed, and the residual ammonia concentration.
        • The ammonia flux was about 1.1 mmol g-1h-1 for all of the ammonia concentrations.
      • At ammonia concentrations above 45 mM, the respiratory quotient (CO2 produced/O2 consumed) remained constant
      • At ammonia concentrations lower than 44 mM, the respiratory quotient varied
        • Overall, there were no significant differences in carbon metabolism when there was an increased amount or an excess of ammonia.
      • Increasing the ammonia concentration can also have an effect on intracellular alpha-ketoglutarate, glutamate, and glutamine concentrations.
        • When ammonia was in excess, the alpha-ketoglutarate concentration decreased from 10 to ~5 μmol g-1, and the glutamate concentration increased from 75 to 220 μmol g-1. The glutamine concentration increased from 4 to 27 μmol g-1.
    • Northern analysis
      • To observe the effect of increasing ammonia concentrations on the RNA levels of nitrogen-regulated genes, northern (RNA) analyses were performed.
      • Amino acid permease-encoding genes: GAP1 and PUT4
      • Biosynthetic genes: ILV5 (alpha-acetoacetate reductoisomerase) and HIS4 (histidinol dehydrogenase)
      • RNA detection
        • P-labelled oligonucleotides used to detect the levels of GDH1, GLN1, GAP1, ILV5, HIS4, ACT1, and H2A-H2B RNA.
        • Another specific oligonucleotide (described further in the study) was used to analyze PUT4 RNA levels.
        • A 32P-labelled 2.5-kb XhoI-BamHI DNA fragment was used to detect GDH2 RNA.
      • X-ray films were used to quantify the data
      • GDH1 RNA was repressed and GDH2 RNA was induced by the concentration of ammonia
      • As ammonia concentrations increase, the amounts of GAP1 and PUT4 RNA gradually decrease. These genes are regulated by the ammonia concentration.
      • ILV5 RNA and HIS4 RNA (amino acid biosynthetic genes) increased as the ammonia concentration increased, but only until about 66 mM.
        • Further increasing ammonia concentrations caused the levels to decrease again.
    • Enzyme Activities
      • Aimed to see if changes in ammonia concentrations let to changes in enzyme activity
        • Enzymes that convert ammonia into glutamate/glutamine
      • Determined the levels of NADPH-glutamate dehydrogenase, NAD-GDH, and GS activity
      • NADPH-GDH
        • When ammonia concentration was increased from 29 to 118 mM, activity decreased from 4.1 to 1.8 μmol min-1 mg-1.
        • Level of GDH1 expression also decreased
      • NAD-GDH
        • When ammonia concentration was increased from 29 to 61 mM, activity increased from 0.01 to 0.15 μmol min-1 mg-1, but further increase in ammonia concentration did not further increase the activity.
      • GS activity
        • When the ammonia concentration was increased up to 61 mM, GS activity decreased slightly. No further changes after 61 mM.
  • Figures
    • Fig. 1A
      • The X axes represents the ammonia concentration (mM)
      • The Y axes on the left represents the residual ammonium concentration (mM), and the Y axes on the right represents the biomass of S. cerevisiae.
      • These measurements were made by growing the yeast in a continuous culture and manipulating the concentration of ammonia in the food source.
      • Trends
        • As ammonia concentration increases, the residual ammonia concentration increases and the biomass slightly increases. There is a point where the biomass stops increasing, indicating that the yeast has reached the carrying capacity of the population.
    • Fig. 1B
      • The X axes represents the ammonia concentration (mM)
      • The Y axes on the left represents the O2 consumption and the CO2 production. The Y axes on the right represents the respiratory quotient (CO2 produced/O2 consumed).
      • These measurements were made by monitoring the CO2 production and the O2 consumption as the ammonia concentration increased.
      • Trends
        • At an ammonia concentration 44 mM or more, the respiratory quotient remains fairly constant, meaning the yeast is producing CO2 and using O2 at a relatively constant rate.
        • At concentrations below 44 mM, the respiratory quotient varies greatly.
    • Fig. 1C
      • This figure displays the how the ammonia concentration affects the concentrations of alpha-ketoglutarate, glutamate, and glutamine.
      • The X axes, which is used for all 3 graphs, represents the concentration of ammonia
      • The Y axes represents the concentration of each individual component.
      • These measurements were made by observing how intracellular ammonia reacts with alpha-ketoglutarate.
      • Trends
        • As the concentration of ammonia increases, the concentration of alpha-ketoglutarate decreases, the concentration of glutamate increases, and the concentration of glutamine increases.
        • More ammonia means that more alpha-ketoglutarate can be converted into glutamate, which can then be converted into glutamine.
    • Fig. 2
      • The X axes represents the ammonia concentration (mM)
      • The Y axes represents the % Expression
      • The left panel displays the expression levels of GDH1 and GDH2. The center panel displays the expression levels of GAP1 and PUT4. The right panel represents the expression levels of GLN1, HIS4, and ILV5.
      • These measurements were made by calculating the intensity ratio between the entire banding of the gene of interest and of the reference gene.
      • Trends
        • GDH1 RNA was repressed and GDH2 RNA was induced by the concentration of ammonia
        • As ammonia concentrations increase, the amounts of GAP1 and PUT4 RNA gradually decrease. These genes are regulated by the ammonia concentration.
        • ILV5 RNA and HIS4 RNA (amino acid biosynthetic genes) increased as the ammonia concentration increased, but only until about 66 mM.
    • Fig. 3
      • The X axes represents the ammonia concentration
      • The Y axes represents the in vitro activity levels of NADPH-GDH, NAD-GDH, GS transferase, and GS.
      • The top panel displays data for NADPH-GDH, the middle panel displays data for NAD-GDH, and the bottom panel displays data for GS transferase and GS activity.
      • These measurements were under Vmax conditions (NADPH-GDH and NAD-GDH) and as shown by Mitchell and Magasanik (GS).
      • Trends
        • When ammonia concentration was increased from 29 to 118 mM, NADPH-GDH activity decreased from 4.1 to 1.8 μmol min-1 mg-1.
        • When ammonia concentration was increased from 29 to 61 mM, NAD-GDH activity increased from 0.01 to 0.15 μmol min-1 mg-1, but further increase in ammonia concentration did not further increase the activity.
        • When the ammonia concentration was increased up to 61 mM, GS activity decreased slightly. No further changes after 61 mM.
  • Overall conclusion
    • The major finding of this paper was that nitrogen metabolism is regulated by the concentration of ammonia on various levels (physiology, transcription, and enzymes)
    • This paper ruled out the possibility of ammonia flux regulating nitrogen metabolism
      • Points to regulation by intra/extracellular concentrations of ammonia or changes in alpha-ketoglutarate, glutamate, or glutamine
  • Future Directions
    • New experiments could be developed in order to see if S. cerevisiae has an ammonia sensor.
      • This sensor has been found in gram-negative bacteria

Acknowledgements

  • I worked with my homework partner Cameron M. Rehmani Seraji face-to-face outside of class and we texted three times.
  • I worked with my homework partner Margaret J. O'Neil. We texted multiple times throughout the week.
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Lauren M. Kelly 23:50, 1 February 2017 (EST)

References

Dahlquist, Kam D. (2017) BIOL398-05/S17:Week 3. Retrieved from http://www.openwetware.org/wiki/BIOL398-05/S17:Week_3 on 31 January 2017.

ter Schure, E. G., Sillje, H. H., Verkleij, A. J., Boonstra, J., & Verrips, C. T. (1995). The concentration of ammonia regulates nitrogen metabolism in Saccharomyces cerevisiae. Journal of bacteriology, 177(22), 6672-6675.

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