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Part 1: Experimenting with different levels of ammonia

  • Saccharomyces cerevisae, a type of yeast, uses ammonia as its primary nitrogen source
    • Past research has shown that ammonia concentration affects growth
    • In this paper, the researches wanted to know if ammonia flux, and not concentration, is the limiting factor
  • Physiological Parameters
    • S. cerevisae was first grown in cultures with different levels of ammonia, but fixed glucose concentrations
    • Increases in the ammonia concentration from 29 to 61 mM showed a change in biomass from 4.9 to 8.2 g/L
    • After 61 mM, however, there was no significant increase in biomass
    • Formula to calculate ammonia flux: [dilution x (input ammonia concentration - residual ammonia concentration)/biomass]
    • At a level above 44 mM, oxygen consumption was equal to carbon dioxide production
  • Ketoglutarate, Glutamate, Glutamine concentrations as ammonia concentrations were increased
    • Ketoglutarate: Decreased
    • Glutamate: Increased
    • Glutamine: Increased
  • Figure 1
    • X axis: NH4 concentration
    • Y axis:
      • A: Residual NH4 concentration
      • B: 02 consumption/CO2 production
      • C: Ketoglutarate, Glutamate, Glutamine concentrations

Part 2: Gene Expression

  • Northern Analyses are RNA analyses that were done to see how the changes in nitrogen affect nitrogen-regulated genes
  • Figure 2 shows gene expression in response to different levels of NH4 concentrations
    • X axis: NH4 concentration
    • Y axis: Gene expression
  • Permease-encoding genes:
    • GAP1
    • PUT4
  • Biosynthetic genes
    • ILV5
    • HIS4
    • GDH1
    • GLN1

Part 3: Enzymes

  • Figure 3 shows enzyme activity at different NH4 concentrations
    • X axis: NH4 concentration
    • Y axis: Enzyme activity
  • Enzymes Studied:
    • NADPH
    • NAD-DGH
    • GS Transferase
  • NADPH activity consistently decreased with increased NH4
  • NAD-GDH activity consistently increased with increased NH4
  • GS Transferase and Synthetase decreased slightly until 60 mM, but then leveled off with increased NH4
  • The conclusion of the paper is that that the nitrogen metabolism of S. cerevisiae is regulated by ammonia concentration
    • The species may have an ammonia sensor

Word Definitions

  1. Flux: "The total amount of a quantity passing through a given surface per unit time."
  2. 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."
  3. Oligonucleotide: "Linear sequence of up to 20 nucleotides joined by phosphodiester bonds."
  4. Gram-negative:"This is a primary characteristic of bacteria that have a cell wall composed of a thin layer of peptidoglycan covered by an outer membrane of lipoprotein and lipopolysaccharide containing endotoxin."
  5. Gram-positive:"This is a primary characteristic of bacteria whose cell wall is composed of a thick layer of peptidologlycan containing teichoic and lipoteichoic acid complexed to the peptidoglycan."
  6. Glutamate:"Major fast excitatory neurotransmitter in the mammalian central nervous system."
  7. Glutamine:"A crystalline amino acid occurring in proteins; important in protein metabolism.One of the 20 amino acids that are commonly found in proteins."
  8. Isomerase:"An enzyme that converts molecules into their positional isomers."
  9. Synthetase:"Enzymes of class 6 in the e classification, catalyse synthesis of molecules, their activity being coupled to the breakdown of a nucleotide triphosphate."
  10. Metabolite:"Any substance produced by metabolism or by a metabolic process."

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