Conor Keith Week 3

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Purpose

  • The purpose of this assignment is to practice analyzing and evaluating primary research. This analysis will be used to discuss the article with my peers.

List of Terms

  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. Common usage in plasma physics is for flux by itself to mean magnetic field flux, unless specified otherwise.
  2. Biomass: The total mass of all living material in a specific area, habitat, or region.
  3. Acetaldehyde: a colourless, flammable liquid used in the manufacture of acetic acid, perfumes, and flavors. It is also an intermediate in the metabolism of alcohol. It has a general narcotic action and also causes irritation of mucous membranes. Large doses may cause death from respiratory paralysis.
  4. Northern analysis: a technique for identifying a specific form of messenger RNA in cells. It uses a gene probe known to match the RNA being sought.
  5. Biosynthetic: the production of substances by processes occurring in living systems
  6. Oligonucleotide: A macromolecule that consists of a short chain of nucleotides (e.g. a short length of DNA or RNA).
  7. Extracellular: situated or occurring outside cells; for example, extracellular fluid is the fluid surrounding cells.
  8. Dehydrogenase: An enzyme that catalyses the removal of hydrogen from a substrate.
  9. Transferase: An enzyme that catalyses the transfer of a functional group from one substance to another.
  10. Metabolite: Any intermediate in a metabolic pathway.

Outline

Main Result

  • The authors of the article sought to determine whether ammonia flux or concentration is the key growth parameter for Saccharomyces cerevisiae. They found that the rate of ammonia assimilation (i.e. flux) was roughly constant over the range of ammonia concentrations they tested. They also found that increased input ammonia concentrations...

Significance

  • This study is significant because the authors find that increasing ammonia concentrations leads to an increase in biomass concentrations at constant ammonia flux. So ammonia concentration can be adjusted in order to maximize yeast yield.

Previous Studies

  • Previous research indicates that ammonia is the best nitrogen source for growth of Saccharomyces cerevisiae, and that nitrogen metabolism is regulated at the levels of RNA expression and enzyme activity. Prior studies that have utilized continuous cultures have noted the influence of ammonia concentration, but the cultures used differed in external ammonia concentration and ammonia flux. This study used cultures with a fixed amount of flux that varied in ammonia concentration, in order to determine the governing parameter of yeast growth.

Methods

  • S. cerevisiae SU32 was grown in continuous cultures (Chemostat)
  • Feeds with ammonia concentrations of 29, 44, 61, 78, 90, 96, 114, and 118 mM were used with a fixed glucose concentration of 100mM and dilution rate of 0.15 h^-1.
  • Changes in biomass in response to increases in ammonia concentration were measured
  • Ammonia flux was calculated using biomass, ammonia concentration and residual ammonia concentration numbers
  • Ammonia flux[math]=[/math][dilution rate[math]\times[/math](input ammonia concentration[math]-[/math]residual ammonia concentration)[math]\div[/math]biomass]
  • Respiratory quotient (CO2 produced/02 consumed) was measured under different levels of ammonia concentration
  • Effects of increasing ammonia concentrations on intracellular [math]\alpha[/math]-ketoglutarate, glutamate and glutamine concentrations were measured
  • Northern blot analyses were used to track changes in RNA levels of nitrogen-regulated genes with increased ammonia concentrations
    • Amino acid permease-encoding genes: GAP1 and PUT4
    • Biosynthetic genes: ILV5 and HIS4
    • RNA levels were detected with P-labeled oligonucleotides
    • H2A-H2B were used to detect GLN1 RNA levels
    • The data from the northern analyses were quantified using X-ray films over different exposure times
  • Expression levels of genes involved in ammonia utilization GDH1, GDH2 and GLN1 were determined to observe their response to amounts of ammonia
  • The influence of ammonia concentration on GAP1 and PUT4 expression was analyzed at constant flux
  • Enzyme activities level response to changes in ammonia concentration were determined
    • Levels of NADPH-glutamate dehydrogenase, NAD-GDH and GS activity were measured
    • Enzyme activities were measured under V_max conditions

Figures

Figure 1

Figure 1A

  • Ammonia concentration (mM) is measured along x-axis
  • Residual ammonia concentration (mM) is measured along left y-axis ([math]\Box[/math]-[math]\Box[/math])
  • Biomass concentrations (gl^-1) is measured along right y-axis
  • Increase in ammonia concentrations in feed from 29 to 61 mM resulted in an increase of biomass from 4.9 to 8.2 gl^-1.
    • Up to ammonia concentrations of 61 mM residual ammonia concentration remains constant at about 0.022 mM
    • Constant residual ammonia concentrations indicate ammonia limitation
  • At ammonia concentrations greater than 61 mM, residual ammonia concentrations increased linearly up to 62 mM with biomass concentration remaining constant
  • At ammonia concentrations greater than 61 mM, glucose was limiting
  • Over entire range of ammonia concentrations ammonia flux remained constant at 1.1 mmol g^-1 h^-1

Figure 1B

  • Ammonia concentration (mM) is measured along x-axis
  • C02 production and 02 consumption measured along right y-axis
  • Respiratory quotient measured along right y-axis
  • At ammonia concentrations above 44 mM, CO2 production and 02 consumption were relatively constant
    • Under ammonia limitation (ammonia concentrations less than or equal to 44 mM) values differed
  • No changes in glucose concentration
  • Indicates that except at 29 mM ammonia in feed, no significant changes in carbon metabolism occurred when ammonia concentration in feed was increased

Figure 1C

  • Side by side comparison of changes in [math]\alpha[/math]-ketoglutarate, glutamate, and glutamine concentrations in response to changes in ammonia concentrations
  • Ammonia concentration (mM) is measured along x-axis
  • [math]\alpha[/math]-ketoglutarate, glutamate, and glutamine concentrations ([math]\mu[/math]molg^-1) measured along y-axis
    • [math]\alpha[/math]-ketoglutarate concentration decreased when conditions changed from ammonia limitation to excess
    • Intracellular glutamate concentration increased from 75 to 220 [math]\mu[/math]molg^-1
    • Intracellular glutamine concentration increased linearly from 4 to 29 mM to 27 [math]\mu[/math]molg^-1 at 118 mM ammonia
  • This indicates intracellular glutamate and glutamine concentration increases with increasing ammonia concentration and constant flux

Figure 2

  • Models RNA expression of nitrogen-regulated genes at different ammonia concentrations in feed
  • Expression levels of ACT1 and H2A-H2B were used as internal controls for amount of RNA loaded
  • Left panel depicts levels of GDH1 and GDH2; central panel GAP1 and PUT4; right panel GLN1 HIS$ and ILV5
  • Quantified by calculating the intensity ratio between entire banding of observed gene and reference gene
  • X-axis measures ammonia concentration (mM)
  • Y-axis measures expression levels of genes at different ammonia concentrations in percentages of expression
  • With increasing ammonia concentrations up to 78 mM levels of GDH1 RNA remained constant, but at higher concentrations levels of GDH1 RNA decreased
    • Max GLN1 at 61 mM
  • Between concentrations of 29 and 44 mM GDH2 RNA could be detected, as concentrations increased to 61 mM, GDH2 RNA levels increased
  • Concentration of ammonia repressed GDH1 and induced GDH2 RNA expression of nitrogen regulated gasses
  • Expression of GAP1 is observed to be regulated in response to ammonia concentration of flux, PUT4 has been shown to be regulated similarly
    • Under constant flux, the two behaved similarly up to 118mM
    • At 118 mM, no GAP1 RNA was detected but PUT4 RNA was still present
  • In ammonia limited environment, a relation between flux, residual ammonia and GAP1 expression was observed
    • In ammonia limited culture with a large excess of glucose, GAP1 did not change with increasing flux
  • From this it is deduced that GAP1 and PUT4 are regulated by ammonia concentration, but not flux
  • RNA amount of amino acid biosynthetic genes ILV5 and HIS4 increased with increasing extracellular ammonia concentrations and had maximum at 66 mM
    • With further increases, levels of ILV5 and HIS4 RNA decreased again
  • Figure 2 shows similarity among expression patterns of Gen4p-regulated genes

Figure 3

  • Measures In vitro activity levels of NADPH-GDH, NAD-GDH, GS transferase and GS at different ammonia concentrations in feed
  • X-axis measures ammonia concentration
  • Y-axis measures NADPH-GDH activity levels in top panel, NAD-GDH activity levels in middle panel, and GS transferase/GS synthetase levels in bottom panel ([math]\mu[/math]molmin^-1mg^-1)
  • Tracks changes in levels of activity of enzymes involved in conversion of ammonia to glutamate or glutamine in response to changed ammonia concentrations
    • NAD-GDH and GS activity were determined to analyze these changes
    • Measured under V_max conditions (maximal activity of enzyme)
  • When input ammonia concentrations increased from 29 to 118 mM, the activity level of NADPH-GDH decreased from 4.1 to 1.8 [math]\mu[/math]molmin^-1mg^-1
  • Decrease in NADPH-GDH was accompanied by decrease in level of GDH1 expression, so decrease can partly be attributed to transcriptional regulation
  • NAD-GDH activity sharply increased between 29 and 61 mM ammonia, but further increase in ammonia concentration did not result any further increase in activity
  • The relation between GDH2 RNA expression and NAD-GDH indicates the enzyme is mainly regulated at the transcriptional level
  • Slight decreases in GS transferase and GS activity were observed with increasing ammonia concentrations up to 61 mM, but further increases did not result in significant changes
    • Altered level of GS activity cannot be explained by altered transcription, so it must be posttranscriptional
  • Changes in NADPH_GDH and GS activity are reflected in ammonia, [math]\alpha[/math]-ketoglutarate, glutamine and glutamate concentrations

Conclusion

  • The data shows that ammonia concentration regulates the nitrogen metabolism of S. cerevisiae on physiological, gene expression, and enzyme levels. Ammonia flux was held constant, so the authors were able to conclude that nitrogen metabolism responses are regulated by wither extracellular or intracellular concentrations of ammonia or by levels of intracellular metabolites. The data indicates that the yeast may have an ammonia sensor, similar to gram-negative bacteria.

Future Research

  • Further research should be performed on the relation between enzyme activities and gene expression. The authors noted that changes in activity of some enzymes can be attributed to transcriptional regulation, but didn't go into any further detail. Further research can also be performed on the implied ammonia sensor in the conclusion of the article.

Acknowledgements

I worked with my homework partner Nika Vafadari. We communicated over facebook. Except for what is noted this assignment was completed by me and not copied from another source.

Conor Keith 00:11, 2 February 2017 (EST)

References

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