Natalie Williams Week 2

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Week 2

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Biological Terms

These are the following terms that I had a hard time fully understanding. I did not know exactly what they meant or what they were in relation to what was done in the article.

  • Proline: a heterocyclic, non-polar amino acid that is present in all proteins studied to date [Source]
  • Continuous Cultures: a technique for production of microbes or microbial products in which nutrients are continuously supplied to the fermenter [Source]
  • Assimilation: The conversion of nutriment into a useable form (e.g. liquid or solid) that is incorporated into the tissues and organs following the processes of digestion [Source, (3)]
  • Permease: protein in bacteria that is responsible for transport of substances into or out of the cell; sometimes referred to as a transport protein [Source]
  • Northern analyses (RNA): also known as Northern blot analysis; a procedure used mostly to separate and identify RNA fragments; typically the RNA fragment is moved to an agarose gel to a nitrocellulose filter [Source]
  • Oligonucleotides: a short polymer of nucleotides [Source]
  • GS (Glutamate synthesase) transferase: An enzyme that catalyses a reaction between ATP, l‐glutamate, and NH3 to form ADP, orthophosphate, and l‐glutamine [Source]
  • α-ketoglutarate: a compound that has major roles in carbohydrate and amino acid metabolism [Reference]
  • Glutamate: major fast excitatory neurotransmitter in the mammalian central nervous system [Source]; a salt of glutamic acid [Reference]
  • Glutamine: an amino acid commonly found in proteins; its the amide at the γ–carboxyl of glutamate [Source]

Outline

Introduction

  • Yeast uses ammonia more than other sources of nitrogen
  • Breakdown of nitrogen is seen on the level of gene expression and enzyme activity
    • The studies have show that the fluctuation of nitrogen influences various cultures rather than the concentration of nitrogen (ammonia)
  • In this study, the yeast all intake and remove ammonia at the same rates but the amount that each culture is exposed to varies by concentration

Physiological parameters

  • The same culture of yeast is used by under these concentrations:
    • A fixed glucose concentration
    • Dilution rate of .15 h-1
  • An increase of ammonia saw an increase in the biomass of the culture
  • However, there was nitrogen limitation in that after increasing beyond 61 mM, the biomass remained the same (8.2 L)
    • As a result, there was leftover ammonia/feed
    • Glucose become limited resource
  • Ammonia going into biomass occurred generally at 1.1 milli moles g-1
  • The levels of CO2 and O2 being produced and consumed respectively saw minimal changes and were nearly constant
  • No significant changes were seen in the breakdown of carbon when the ammonia concentration switched from limited to excess
  • As the ammonia concentration increases the cells increases the production and concentration of glutamine and the glutamate

Northern Analysis

  • These tests were used to see if the increase in nitrogen had any effect on genes
  • The responses of genes that intake ammonia were observed and recorded to see how they would react to the varying concentrations
  • For specific genes, the gene was either repressed (GDH1) or stimulated (GDH2)

Enzyme Activity

  • NADPH-glutamate dehydrogenase, NAD-GDH, and GS activity were recorded to see how they reacted to the changes in ammonia concentrations
  • When ammonia concentrations increased from 29 to 118 mM, the activity of NADPH-GDH decreased
    • Following this decline, the production of GDH1 fell as well
  • There was a sharp increase in the level of NAD-GDH between 29 and 61 mM, but as the concentrations increased, no change was seen
    • Both of these results suggested that the RNA expression of the affected proteins as well as the electron carrier transporters were controlled at a transcriptional level
  • GS transferase and GS activity has an inverted relationship with the concentration of ammonia up to 61 mM
  • However, GS activity did not seem to be affected by transcription, so it must have posttranscriptional alterations.
  • NADPH and GS activity affect ammonia, α-ketoglutarate, glutamate, and glutamine concentrations

Conclusions

  • This data shows that ammonia concentration affects nitrogen metabolism on the transcriptional and enzymatic levels in yeast
  • Nitrogen metabolism can be affected either by intracellular or extracellular ammonia concentrations or by altering glutamate, glutamite, and α-ketoglutarate levels


What is the main result presented in this paper?

  • The main result was that increased ammonia concentration extracellularly had a positive relationship with the activity of intracellular glutamine and glutamate (enzyme activity) and generally activating impact on gene regulation.

What is the importance or significance of this work?

  • The importance of this work further explores and discusses how a eukaryotic organism uses its nitrogen metabolism. By studying this further, we have a better understanding of how nitrogen metabolism can affect our bodies and the organisms around us. It shows us that nitrogen affects various levels of the cell from transcription to enzyme activity.

What were the limitations in previous studies that led them to perform this work?

  • Previously, the researchers did not seem to know what really affects nitrogen metabolism but what the ammonia concentration affects. Previous research also suggested that if it is the first (controlled by ammonia concentrations), then yeast would have to have an ammonia sensor that helps it react to the nitrogen concentrations in its environment

What were the methods used in the study?

  • One of the methods was to control the ammonia concentrations for various cultures. Analysis of the oxygen consumption and carbon dioxide production was used so that the researchers could see if ammonia concentration influenced the cell's more macro-scale functions. The Northern blot analysis was used to look at the specific genes that nitrogen concentrations would affect.

What is the overall conclusion of the study and what are some future directions for research?

  • The overall conclusion was that ammonia concentrations greatly influences the functions of different proteins, enzymes, gene expression amongst other activities in yeast. For future research, one may want to see how the yeast receives signals that the ammonia has changed.

Figures

A

  • X: NH4 concentration (mM)
  • Y axes: Residual NH4 concentration (mM)
    • Biomass (g-1)
    • NH4 flux
  • Can see that the NH4 flux was nearly constant throughout the changing of NH4 concentrations; 1mmol/(gxh)
  • At 60 mM there was no ammonia left, but as the concentration increased, the residual NH4 increased almost linearly
  • There was a sharp increase, 4 to 8, in the biomass from 20 – 60 mM of NH4 and the increase of the NH4 after 60mM just caused a fluctuation between 7 and 9.

From this figure, we can see that increasing the ammonia concentration impacts the growth rate initially, but after a certain concentration, no significant change can be seen.
B

  • X: NH4 concentration (mM)
  • Y axes: O2 consumption (mmol/gh)
    • CO2 production (mmol/gh)
    • Respiratory Quotient
  • When there are low concentrations of ammonia, O2 is consumed until there is at least 40mM of NH4.
  • However, for CO2, its production decreases at low concentrations of ammonia. When it reaches ~60mM of NH4, the levels out and fluctuates around 4.5.
  • The respiratory quotient shows change when both the O2 and CO2 levels change; yet, when O2 begins to level off, the quotient stabilizes.

C
Each of the graphs shows the level of their respective amino acids: Glutamate, glutamite, and α-ketoglutarate

  • X: NH4 concentration (mM) for each of the graphs
  • Y: α-ketoglutarate (micromol/g)
    • Glutamate (micromol/g)
    • Glutamine (micromol/g)
  • For α-ketoglutarate, the graph shows that its levels decrease as the concentration of NH4 increases until it fluctuates at 60 mM.
  • Glutamate seems to be constant until ~45 mM NH4, where it jumps and continues to increase as the ammonia concentration grows.
  • The glutamine appears to climb linearly as the concentration of ammonia also increases.

One can conclude that the general trend is that there is a positive relationship among these amino acids while α-ketoglutarate has an inverted relationship. Plus, ammonia concentrations affect intracellular metabolisms.
Northern Analysis

  • X: NH4 concentration (mM)
  • Y: Percent expression
  • The left panel had GDH1 and GDH2 depicted
    • GDH1 had a general decline as the concentration of ammonia increased.
    • GDH2 had a varied data set, but the trend seemed to be an increased when compared to initial concentration.
  • The central panel had GAP1 and PUT4. Both seemed to have started with the same level of expression
    • GAP1 saw a slight increase in expression, but after ~45 mM concentration of NH4, its expression level fell.
    • PUT4’s expression increased sharply until the NH4 concentration reached ~45 mM, and then proceeded to plummet. However, its expression remained above GAP1’s.
  • The right paneled shows the expression of GLN1, HIS4, and ILV5.
    • GLN1’s expression rose linearly until it reached its peak at 60 mM before declining.
    • HIS4 had varied expression levels with various peaks. The highest level of its expression came when the ammonia concentration was around 80 mM.
    • The expression of ILV5 increased slowly before falling at an ammonia concentration around 60 mM.

The gene expression of each of these genes seemed to lower overall, except GDH2 and GLN1 whose expressions increased compared to their starting expressions.
Enzyme activities
Top Graph

  • X: NH4 concentration (mM)
  • Y: NADPH-GDH (micromol/(min*g))
  • As the concenctration of ammonia increases in the cell, the activity of NADPH-GDH decreased.

Middle Graph

  • X: NH4 concentration (mM)
  • Y:NAD-GDH(micromol/(min*g))
  • NAD-GDH activity increased as the ammonia concentration increased. They have a positive relationship. There was a decrease when the concentration level reach 80 mM, but after that trough, the activity increased again.

Bottom Graph

  • X: NH4 concentration (mM)
  • Y: GS transferase (micromol/min*g)
    • GS synthetase (micromol/min*g)
  • The GS transferase activity decreases from its initial production until an ammonia concentration of 60 mM, where it stabilized and fluctuated between .57 and .6 micromol/(min*g).
  • Activity of GS synthetase decreased initially as the ammonia concentration increased. However, around 40 mM of ammonia, the GS synthetase reaches an equilibrium point where it wavers between .12 and .9 micromol/(min*g).


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