Alyssa N Gomes Week 2 Journal

Week 2 Journal

Biological Terms

• Permease: A membrane protein that increases permeability of the plasma membrane to a particular molecule, without requiring metabolic energy [Source]
• Proline: One of the 20 amino acids directly coded for in proteins, side chain is bonded to the nitrogen of the amino group as well as the carbon [Souce]
• Gram-negative: Of, or relating to, the group of bacteria that take the color of the counterstain under the microscope following gram-staining. Allows viewer to see morphology of bacteria better [Source]
• Dehydrogenase: Enzyme that oxidizes a substrate by transferring hydrogen to an acceptor that is either NAD/NADP or a flavin enzyme. Helps produce ATP [Source]
• Residual: Remaining or left behind [Source]
• Glutamine: A crystalline amino acid occurring in proteins, important in protein metabolism [Source]
• Transferase: A suffix to the name of the enzyme indicating that it transfers a specific grouping from one molecule to another [Source]
• Posttranscriptional processing: The enzymatic processing of the primary rNA transcript, which produces messenger RNA and transfer RNA [Source]
• Oligonucleotides: Polymers made up of a few (2-20) nucleotides, a short sequence synthesized to match a region where a mutation is known to occur and then used as a probe [Source]
• In Vitro: Within a glass, observable in a test tube, in an artificial environment [Source]

Outline

• Overview
• This experiment was conducted in order to determine whether nitrogen metabolism is due to ammonia concentration or flux.
• This was done by experimenting with nitrogen values, from limited to excess,
• Introduction
  • Ammonia is a preferred nitrogen source for Saccharomyces cerevisiae
  • Nitrogen levels are seen in gene expression and enzyme activity
  • This experiment keeps flux stable and changes the parameters of ammonia concentration
• Physiological Parameters
  • S cerevisiae SU32 was cultured with ammonia concentrations of 29, 44, 61, 66, 78, 90, 96, 114 and 116 mM.
  • Glucose concentration was fixed at 100 mM
  • Dilution rate was 0.15 h-1.
  • Testing
    • Changing the ammonia concentration from 29 to 61 mM led to increase in biomass, from 4.9 g liter-1 to 8.2 liter-1
      • Residual ammonia concentration in the culture medium remained at 0.022 mM
    • Changing the ammonia concentration to above 61 mM led to approx, stable biomass at 8.2 liter-1
      • Residual ammonia concentration increase linearly up to 62 mM.
      • Glucose became limiting
    • Flux was calculated by getting the difference of the input ammonia concentration and the residual concentration multiplied by the dilution rate, then divided by the biomass.
      • Calculating the flux for the entirety of ammonia concentrations, we can see 1.1 mmol g-1 h-1
    • Ammonia concentrations above 44 mM showed stable respiratory quotient (amount of CO2 produced divided by the amount of O2 consumed)
    • No changes in the residual glucose concentration were observed.
    • No significant changes in carbon metabolism except for at 29 mM concentration of ammonia.
  • Intracellular ammonia reacts with α-ketoglutarate to produce glutamate, which then produces glutamine with the addition of an ammonium ion.
    • Increasing the ammonia concentrations showed α-ketoglutarate concentration decreased from 10 to 5 mmol g-1
    • Increasing ammonia concentrations showed intracellular glutamate concentration increased from 75 to 220 mmol g-1
    • Increasing ammonia concentrations showed an intracellular glutamine concentration increase linearly from about 4 mmol g-1 to about 27 mmol g-1

• Northern Analyses
  • Want to see if RNA levels of nitrogen change with increasing/decreasing ammonia concentrations
  • To do this, we compare amino acid permease-encoding genes (GAP1 & PUT4) and biosynthetic genes (ILV5 & HIS4)
    • GDH1, GLN1, GAP1, ILV5, HIS4, ACT1, and H2A-H2B RNA levels were determined with 32P-labelled oligonucleotides
    • PUT4 RNA levels were determined with the oligonucleotide 5'-CTCCTCCTTCTTGGTGTCGCCGCCGCTACC-39
    • GDH2 RNA levels were determined with a 32P-labelled 2.5-kb XhoI-BamHI DNA fragment
  • Quantification of GAP1, PUT4, ILV5, HIS4, GDH1, and GDH2 RNA was done with ACT1 RNA
  • Quantification of GLN1 RNA levels was done with H2A-H2B.
  • Testing on GDH1, GDH2
    • As ammonia concentrations increased, before 78 mM, GDH1 RNA was constant, but after 78 mM, further increases decreased the RNA levels.
    • For GDH2 between ammonia concentrations of 29-44, no RNA was detected. As concentrations increased to 61 mM RNA levels increased.
    • GDH1 RNA levels were maximized at 61 mM
  • Testing on GAP1 and PUT4
    • It was prior been demonstrated that GAP1 and the proline permease Put4pis regulated according to flux or ammonia concentration.
    • Between 29-44 mM levels of ammonia concentration, GAP1 and PUT4 levels were constant.
    • After 44 mM, levels of both GAP1 and PUT4 decreased.
    • At 118 mM, almost no GAP1 RNA was present, but PUT4 was still clearly present.
    • With a small amount of excess glucose, there was a relation between flux, concentration of both RNA and ammonia. Any large excess made no relation.
  • With an increased level of Gcn4p leads to transcription of biosynthetic genes due to amino acid starvation
    • ILV5 and HIS4 RNA amounts increased as ammonia concentrations increased, maximizing at 66 mM. Beyond 66 mM, the RNA amounts for both decreased.

• Enzyme Activities
  • Try to determine whether ammonia concentrations affect the activity levels of enzymes used to convert ammonia to glutamate to glutamine.
    • Determine levels of NADPH-glutamate dehydrogenase, NAD-GDH and GS activity, under Vmax conditions.
    • Increasing ammonia concentrations decreased the activity of NADPH-GDH from 4.1 μmol min-1 mg-1 to 1.8 μmol min-1 mg-1
    • The level of GDH1 expression decreased when ammonia concentrations increased.
    • Decreasing NADPH-GDH, decreased the level of GDH1 expression. As ammonia concentration increased between 29-61 NADPH-GDH increased sharply.
    • As ammonia concentrations increased up to 61 mM, there was slight decrease in GS transferase activity.

• Conclusion
  • The levels of ammonia concentration control the nitrogen metabolism in the S. cerevisiae yeast.
  • Flux remained stable as concentration varied from 29-116 mM
  • Nitrogen metabolism levels varied with concentrations of ammonia or changes in a-ketoglutarate, glutamate, and glutamine