Cameron M. Rehmani Seraji Week 3

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Electronic Lab Notebook Week 3

Purpose

  • The purpose of the week 3 assignment is to prepare for the in class journal club session by defining biological terms we did not know, reading and outlining the paper, and ensuring that all aspects of the paper are understood so the paper can be articulately discussed in class.

Part 1

  • Biological Terms
  1. Ammonia- The common Name for NH3, a strongly basic, irritating, colorless gas which is lighter than air and readily soluble in water. It is formed in nature as a by-product of protein metabolism in animals.
  2. α-ketoglutarate- A non preferred (though often used) name for 2‐oxoglutarate; 2‐oxo‐1,5‐pentanedioate; a compound that has important roles in carbohydrate and amino‐acid metabolism, especially in transamination reactions and as a component of the tricarboxylic acid cycle.
  3. Biosynthetic- The production of a complex chemical compound from simpler precursors in a living organism, usually involving enzymes and energy source.
  4. Flux- The total amount of a quantity passing through a given surface per unit time. Typical quantities include (magnetic) field lines, particles, heat, energy, mass of fluid, etc.
  5. Glutamate- Major fast excitatory neurotransmitter in the mammalian central nervous system.
  6. Glutamine- A crystalline amino acid occurring in proteins; important in protein metabolism.One of the 20 amino acids that are commonly found in proteins.
  7. Metabolism- The process involving a set of chemical reactions that modifies a molecule into another for storage, or for immediate use in another reaction or as a by product.
  8. Permease- General term for a membrane protein that increases the permeability of the plasma membrane to a particular molecule, by a process not requiring metabolic energy.
  9. 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. 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 characterise the binding site of SH3 domains. An amino acid that is found in many proteins (especially collagen).
  10. Sacchromyces cerevisiae- The entire genome of this species has been base sequenced and it is used to do research on the basic cellular mechanics of replication, recombination, cell division and metabolism. It is also economically important in the food industry, where it is used to ferment grain sugars to make beer and as baker's yeast for baking bread or making other food which requires rising by gas bubbles of carbon dioxide. It is also sometimes taken as a vitamin supplement for protein, the B vitamins, and folic acid.

Part 2

Outline

  • Main result presented
    • Nitrogen metabolism in Sacchromyces cerevisiae can be regulated by ammonia concentration in the food source.
    • The ammonia flux is not the governing factor of nitrogen metabolism.
  • Importance or significance of this work
    • Sacchromyces cerevisiae is used to make bread, beer, and other food products so understanding the effect of ammonia concentration on it's nitrogen metabolism can help create more efficient ways to grow this type of yeast.
  • Limitations in previous studies that led them to perform this work
    • Previous studies determined that ammonia concentration was the main factor on nitrogen metabolism in S. cervisiae being grown in continuous cultures. However, these studies did not account for the ammonia flux as a factor for nitrogen metabolism.
  • Methods used in the study
    • Physiological Parameters
      • S. cerevisiae SU 32 was grown in continuous cultures with feeds that had different ammonia concentrations of 29, 44, 61, 66, 78, 90, 96, 114, and 118 mM. There was also a fixed glucose concentration of 100 mM in the medium and a dilution rate of 0.15 h^-1.
      • The change in biomass was calculated as the concentration of ammonia was increased in order to see the effect ammonia concentration had on biomass.
      • Biomass increased from 4.9 to 8.2 g/liter as the ammonia concentration increased from 29 to 61 mM. The biomass remained constant at about 8.2 g/liter and glucose became limiting at ammonia concentrations higher than 61 mM.
      • The ammonia flux was calculated over the entire range of ammonia concentration.
        • The ammonia flux into biomass remained at 1.1 mmol g^-1 h^-1.
      • The respiratory quotient remained relatively constant when the input ammonia concentration was above 44 mM, but when input ammonia concentration was lower than 44 mM, the value of the respiratory quotient did not remain constant.
      • The concentrations of α-ketoglutarate, glutamate, and glutamine are affected by an increase in ammonia concentration.
        • As the ammonia concentration increased, the concentration of α-ketoglutarate decreased from 10 to 5 μmol g^-1, the concentration of glutamate increased from 75 to 220 μmol g^-1, and the concentration of glutamine increased from 4 to 27 μmol g^-1.
    • Northern Analyses
      • The focus of this section is to observe what effects an increase in ammonia concentration will have on the RNA levels of nitrogen-regulated genes.
      • GAP1 and PUT4 are the two amino acid permease-encoding genes used and ILV5 and HIS4 were the two biosynthetic genes used.
      • A set of P-labelled oligonucleotides were used to detect the levels of GDH1, GLN1, GAP1, ILV5, HIS4, ACT1, and H2A-H2B RNA. GDH2 RNA was detected using a 32P-labelled 2.5-kb Xhol-BamHI DNA fragment.
      • X-ray films were used to quantify the data and it was determined that the concentration of ammonia induced GDH2 RNA and repressed GDH1 RNA.
      • The amounts of GAP1 and PUT4 gradually decreased as the ammonia concentration increased.
      • The amounts of ILV5 and HIS4 increased as the ammonia concentration increased, but started to decrease once the ammonia concentration increased about 66 mM.
    • Enzyme Activites
      • Look to see if the enzyme activity of enzymes that convert ammonia into glutamate and glutamine are affected by the change in ammonia concentration.
      • The enzymes investigated were NADPH-glutamate dehydrogenase (GDH), NAD-GDH, and GS transferase and GS activity.
      • NADPH-GDH
        • The activity of NADPH-GDH decreased from 4.1 to 1.8 μmol min^-1 mg^-1 as the concentration of ammonia increased from 29 to 118 mM.
        • GDH1 expression decreased.
      • NAD-GDH
        • The activity of NAD-GDH increased 0.01 to 0.15 μmol min^-1 mg^-1 as the concentration of ammonia increased from 29 to 61 mM.
        • Further increase in ammonia concentration did not further increase the activity.
      • GS transferase and GS activity
        • The levels of GS transferase decreased from 0.82 to 0.60 μmol min^-1 mg^-1 and GS activity decreased from 0.15 to 0.10 μmol min^-1 mg^-1 were observed as the ammonia concentration increased to 61 mM, but there was no further change after that concentration.
  • Result shown in each of the figures.
    • Figure 1A. An increase in ammonia concentration lead to an increase of biomass. The ammonium concentration remained at 0.022 mM and the residual ammonia concentration increased linearly from 61 mM to 62 mM and the biomass remained at 8.2 g/liter.
    • Figure 1B. CO2 produced and O2 consumed remain relatively constant when the input ammonia concentration is greater than 44 mM. CO2 produced/O2 consumed equals the respiratory constant in the system. However, when equal to or lower than 44 mM, the system is under ammonia limitation and the amount of CO2 produced is different from O2 consumed. This data indicates that no significant changes in carbon metabolism occurred when ammonia concentration increased from being limited to being in excess.
    • Figure 1C. The α-ketoglutarate concentration decreased as the ammonia concentration in the food changed from being limited to being in excess. In contrast to α-ketoglutarate, the intracellular glutamate concentration increased as the ammonia concentration changed from ammonia limited to ammonia excess. Similar to intracellular glutamate, the intracellular glutamine concentration increased as the ammonia concentration in the food increased. Thus, with constant increase of ammonia concentration, α-ketoglutarate concentration decreased and intracellular glutamate and glutamine concentrations increased.
    • Figure 2. The levels of RNA expression of nitrogen regulated genes: GAP1, PUT4, GDH1, GDH2, GLN1, HIS4, AND ILV5. The percent expressions of GAP1, PUT4, GDH1, GLN1, and HIS4 all decreased as the concentration of ammonia increased. In addition, the percent expressions of GDH2 and ILV5 both increased as the concentration of ammonia increased.
    • Figure 3. The concentration of NADPH-GDH, GS transferase, and GS synthetase all decreased as the concentration of ammonia increased. Conversely, the concentration of NAD-GDH increased as the concentration of ammonia increased.
    • X and Y axes
      • Figure 1A. The X axis represent the ammonia concentration (mM) and the Y axis represents the residual ammonia concentration and biomass
      • Figure 1B. The X axis represents the ammonia concentration (mM) and the Y axis represents the respiratory quotient (CO2 produced/O2 consumed).
      • Figure 1C. The X axis represents the ammonia concentration (mM) and the Y axis for each graph represents the concentrations of α-ketoglutarate, intracellular glutamate, and intracellular glutamine, respectively for the three graphs.
      • Figure 2. The X axis represents the ammonia concentration (mM) and the Y axis represents the percent expression of the seven different nitrogen-regulated genes.
      • Figure 3. The X axis represents the ammonia concentration (mM) and the Y axis represents the concentrations of NADPH-GDH, NAD-GDH, and GS transferase and Gs synthetase, respectively for the three graphs.
    • How the measurements were made
      • Figure 1A. The measurements were made by changing the concentration of ammonia in the food source for growing Sacchromyces cerevisiae.
      • Figure 1B. The measurements were made by monitoring the respiratory quoteint (CO2 produced/ O2 consumed) as ammonia concentration increased.
      • Figure 1C. The measurements were made by monitoring the concentration of α-ketoglutarate, glutamate, and glutamine as the ammonia concentration increased.
      • Figure 2. The measurements were made by calculating the percent expression as the ammonia concentration increases. The percent expression is completed by comparing the intensity ratio between the gene of interest and a reference gene.
      • Figure 3. The measurements were made by calculating the levels of NADPH-GDH, NAD-GDH, and GS. The NADPH-GDH and NAD-GDH were measured under Vmax conditions and GS was measured the same way as Mitchell and Magasanik.
    • Trends are shown by the plots and what conclusions can you draw from the data
      • Figure 1A. The system was limited by ammonia and that glucose becomes limiting in conditions with an ammonia concentration greater than 61 mM.
      • Figure 1B. There was no change in residual glucose concentration, therefore no significant changes in carbon metabolism occurred when ammonia concentration increased from being limited to being in excess.
      • Figure 1C. With the constant increase of ammonia in the food, α-ketoglutarate concentration decreased and intracellular glutamate and glutamine concentrations increased.
      • Figure 2. RNA expression of nitrogen-regulated genes was repressed (GAP1, PUT4, GDH1, GLN1, and HIS4) and induced (GDH2 and ILV5) by the concentration of ammonia.
      • Figure 3. The change in NADPH-GDH, GS transferase, and GS synthetase activity levels affect the ammonia, α-ketoglutarate, glutamate, and glutamine concentrations are
  • Overall conclusion of the study
    • The overall conclusion of the study is the change in ammonia concentration in food can affect the the nitrogen metabolism of Sacchromyces cerevisiae.
    • Shows that the nitrogen metabolism is not regulated by ammonia flux because in this experiment the ammonia flux remained constant.
  • Future Directions for Research
    • Further experiments could be completed to confirm if S. cerevisiae has an ammonia sensor.
      • Similar to the two-component sensing system for nitrogen found in gram-negative bacteria

Acknowledgement

  • I worked with my homework partner Lauren M. Kelly face-to-face outside of class.
  • I worked with my homework partner Margaret J. O'Neil and Lauren M. Kelly. 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.

References

  1. Ammonia- http://www.biology-online.org/dictionary/Ammonia
  2. α-ketoglutarate- http://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-10709?rskey=dh3BXa&result=1
  3. Biosynthetic- http://www.biology-online.org/dictionary/Biosynthesis
  4. Flux- http://www.biology-online.org/dictionary/Flux
  5. Glutamate- http://www.biology-online.org/dictionary/Glutamate
  6. Glutamine- http://www.biology-online.org/dictionary/Glutamine
  7. Metabolism- http://www.biology-online.org/dictionary/Metabolism
  8. Permease- http://www.biology-online.org/dictionary/Permease
  9. Proline- http://www.biology-online.org/dictionary/Proline
  10. Sacchromyces cerevisiae- http://www.biology-online.org/dictionary/Saccharomyces_cerevisiae


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