Kevin Matthew McKay week 3

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===Enzyme activities===
===Enzyme activities===
*Levels of NADPH-glutamate dehydrogenase, NAD-GDH, and GS activity were determined against different concentrations of ammonia. Figure 3
*Levels of NADPH-glutamate dehydrogenase, NAD-GDH, and GS activity were determined against different concentrations of ammonia. Figure 3
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**As ammonia increased from 29 to 118 mM, activity of NADPH-GDH decreased from 4.1 to 1.8 micromoles/min*mg (as shown in figure 3 top panel x axis ammonia co8ncentration y axis enzyme)
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**As ammonia increased from 29 to 118 mM, activity of NADPH-GDH decreased from 4.1 to 1.8 micromoles/min*mg (as shown in figure 3 top panel x axis ammonia concentration y axis enzyme)
**This was accompanied by decrease in level of GDH1 expression so NADPH-GDH decrease could have been partly transcriptional.
**This was accompanied by decrease in level of GDH1 expression so NADPH-GDH decrease could have been partly transcriptional.
**NAD-GDH activity increased drastically between 29 and 61 mM ammonia, but further ammonia increase yielded no change overall (middle panel, ammonia concentration x axis)
**NAD-GDH activity increased drastically between 29 and 61 mM ammonia, but further ammonia increase yielded no change overall (middle panel, ammonia concentration x axis)

Current revision

BIOL398-03/S13:Week 3

Contents

Definitions

  • (I could not find words that I did not know the definition of so I listed 10 words I thought most difficult)
  • Biosynthetic-relating to or produced by biosynthesis:The production of a complex chemical compound from simpler precursors in a living organism, usually involving enzymes (to catalyze the reaction) and energy source (such as ATP)-[[1]]
  • 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. [[2]]
  • Oligonucleotides-polymers made up of a few (2-20) nucleotides. In molecular genetics, they refer to a short sequence synthesised to match a region where a mutation is known to occur, and then used as a probe (oligonucleotide probes). [[3]]
  • arbitrary-Depending on will or discretion; not governed by any fixed rules; [[4]]
  • gram-negative bacteria- bacteria which lose crystal violet stain but are stained pink when treated by grams method. [[5]]
  • glutamate- major fast excitatory neurotransmitter in the mammalian central nervous system. [[6]]
  • dilution-In microbiologic techniques, a method for counting the number of viable cells in a suspension; a sample is diluted to the point where an aliquot, when plated, yields a countable number of separate colonies. [[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.glutamine [[8]]
  • biomass-The total mass of all living material in a specific area, habitat, or region. [[9]]
  • northern blot- An electroblotting method in which rNA is transferred to a filter and detected by hybridisation to (32)P labelled RNA or dNA. [[10]]


The Concentration of Ammonia Regulates Nitrogen Metabolism in Saccharomyces Cerevisiae

Experiment

  • Saccharomyces Cerevisae (Yeast) was grown in culture with varying levels of nutrient ammonia.
    • This species of yeast prefers ammonia as its source of nitrogen
  • While the ammonia input rate was dynamic, the rate at which it was taken up and converted into usable form was constant (flux).
    • Flux was (dilution rate x (input ammonia concentration – residual concentration)/ biomass)
  • So this study was focused on level of concentration (it being the variable parameter).
    • Different concentrations influenced gene expression and enzyme activities differently
  • Increase in ammonia concentration resulted in an increase of biomass in culture
    • Figure 1(a) Showed as ammonia concentration increased (x axis), residual ammonia (left y axis) and Biomass increased (right y axis)
    • After an increase to 61mM of ammonia, glucose became the limiting nutrient (scarce resource of the yeast)
    • Above level of 44mM ammonia, ratio of CO2 produced to O2 consumed remained constant as shown in figure 1(b)-y axis is ratio while x axis is ammonia concentration
    • However when ammonia was limited (below 44mM), ratio differed, while no changes in residual glucose concentration was observed
      • This indicates that except at 29mM ammonia concentration, metabolism in yeast was unchanged when concentration was increased
  • Ammonia in the yeast is used in a pathway that leads to glutamine production (an amino acid used to build proteins), this process was analyzed by method of ter Shure et al.
  • Figure 1(c): as culture changed from ammonia limitation concentration to excess (x axis), alpha ketoglutarate concentration decreased (y axis)
    • Change from low to high concentration of ammonia caused increase in intracellular glutamate concentration (y axis) and increase in intracellular glutamine concentration (y axis)
  • All three graphs included constant ammonia flux while increasing concentration of ammonia

Gene Expression Level

  • Northern Blots were used to see whether altering levels of ammonia concentration affected nitrogen regulated genes (RNA levels)
    • RNA levels were detected by P32 labeled oligonucleotides
    • Expression levels of genes that utilize ammonia (GDH1, GDH2, GLN1 RNA levels) were studied with different concentrations of ammonia
  • Figure 2
    • left panel shows that as ammonia concentration increased to 78mM (x axis), RNA remained constant (GDH1), but after that, RNA level decreased (y axis)
    • max expression of GLN1 occurred at 61mM (right panel).
    • At levels of 29 and 44 mM, no RNA (GDH2) could even be detected. But increase in GHD2 could be detected at 61mM ammonia
    • Increased ammonia repressed expression in GDH1 and increased expression in GDH2.
    • Change in ammonia concentration or flux regulates levels of GAP1 and PUT4 gene
      • They were analyzed however, under constant ammonia flux
    • From 29-44mM ammonia (central panel) , expression was constant, but after 44, expression or RNA levels decreased.
    • At 118 mM ammonia, PUT4 RNA was still present but GAP 1 was absent
    • When ammonia was limited, GAP1 expression did not change with increasing ammonia flux, so it and PUT4 are not regulated by flux
    • Amino acid starvation caused increased level of Gen4P transcription regulator
    • Gene expression of genes ILV5 and HIS4 increase with increasing extracellular ammonia concentration topping out at 66mM and decreasing subsequently (right panel)

Enzyme activities

  • Levels of NADPH-glutamate dehydrogenase, NAD-GDH, and GS activity were determined against different concentrations of ammonia. Figure 3
    • As ammonia increased from 29 to 118 mM, activity of NADPH-GDH decreased from 4.1 to 1.8 micromoles/min*mg (as shown in figure 3 top panel x axis ammonia concentration y axis enzyme)
    • This was accompanied by decrease in level of GDH1 expression so NADPH-GDH decrease could have been partly transcriptional.
    • NAD-GDH activity increased drastically between 29 and 61 mM ammonia, but further ammonia increase yielded no change overall (middle panel, ammonia concentration x axis)
      • GDH2 RNA expression suggests that this enzyme is regulated at the level of transcription
    • Slight decrease in level of GS activity were seen as ammonia concentrations increased to 61mM but activity level was stagnant beyond that point (bottom panel, ammonia concentration x axis)
      • This activity was not regulated by transcription
  • Experiment shows that concentration of nitrogen (not flux) regulates metabolism of S. cerevisiae
  • This could mean S. cerevisiae has an ammonia sensor like those found in gram- negative bacteria
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