Tessa A. Morris Week 2

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User Page: Tessa A. Morris
Course Page: Biomathematical Modeling

Assignment

Week 2 Assignment

Ten Biological Terms

  1. Flux: (Science: radiobiology) 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. Source
  2. Northern Blot Analysis: A procedure... used mostly to separate and identify rNA fragments; typically via transferring RNA fragments from an agarose gel to a nitrocellulose filter followed by detection with a suitable probe. Source
  3. 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) Source
  4. 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; The chemical alteration of substances in the bloodstream by the liver or cellular secretions. Source
  5. Dehydrogenase:enzyme that oxidizes a substrate by transferring hydrogen to an acceptor that is either NAD/NADP or a flavin enzyme. An enzyme that is used to remove hydrogen from its substrate, which is used in the cytochrome (hydrogen carrier) system in respiration to produce a net gain of ATP. Source
  6. 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. A type of protein believed to be involved in active transport and acts as a protein carrier. Source
  7. Metabolite: a chemical compound that is produced or consumed during metabolism. Source
  8. Metabolism: the sum of all the physical and chemical processes by which living cells produce and maintain themselves. Source
  9. Synthetase: catalyse synthesis of molecules, their activity being coupled to the breakdown of a nucleotide triphosphate. Source
  10. Transferases: enzymes that catalyze the transfer of functional groups between donor and acceptor molecules. Source

Outline

I. Overview
A. The purpose was to investigate if the governing factor of nitrogen metabolism may be the concentration of ammonia rather than its flux
B. Saccharomyce cerevisiae was the model organism
C. The effect of the input of ammonia concentrations ranged from nitrogen limitation to nitrogen excess and glucose limitation
II. Introduction
A. Ammonia is a preferred nitrogen source for Saccharomyces cerevisiae
B. The components of nitrogen metabolism are regulated at both the level of gene expression and the level of enzyme activity
C. There is evidence saying that ammonia concentration itself plays a part in nitrogen metabolism
1. Cultures differ in the external ammonia concentration and in the rate of ammonia assimilation
2. Because of the varying parameters it is possible that the flux, rather than the concentration is the governing parameter
III. Physiological parameters
A. S. cerevisae SU32 was grown in continuous cultures with feeds containing the following ammonia concentrations: 29, 44, 61, 66, 78, 90, 96, 114, and 118 mM
B. The glucose concentration was fixed at 100 mM
C. The dilution rate was held constant at 0.15 h-1
IV. Data
A. Increasing the ammonia concentration from 29 to 61 mM
1. Biomass increased from 4.9 to 8.2 g liter-1
2. The residual ammonia concentration in the culture medium was constant at about 0.022 mM
3. Ammonia was limiting
B. When the ammonia concentration was above 61 mM
1. Biomass remained at about 8.2 g
2. The residual ammonia concentration in the culture medium increased linearly up to 62 mM
3. Glucose became limiting
C. The ammonia flux was calculated by taking the difference between the input ammonia concentration and the residual ammonia concentration, dividing by the biomass, and then multiplying by the dilution rate
1. Over the entire range of ammonia concentrations, the ammonia flux into biomass was about 1.1. mmol g-1 h-1
D. At an input ammonia concentration above 44 mM, the CO2 production and O2 consumption, thus the respiratory quotient (CO2 produced divided by O2 consumed), remained relatively constant
1. Less than or equal 44 mM the values for CO2 production and O2 consumption differed, but no no changes in the residual glucose concentration were observed
E. Data showed that except at 29 mM ammonia in the feed, no significant changes in the carbon metabolism occurred when the ammonia concentration in the feed was increased and the culture was switched from ammonia limitation to ammonia excess
V. Chemical Process
A. Intracellular ammonia reacts with α-ketoglutarate to produce glutamate
B. Glutamate is converted into glutamine by incorporation of another ammonium ion
VI. Effect of increasing ammonia concentrations on the intracellular α-ketoglutarate, glutamate, and glutamine concentrations
A. α-ketoglutarate concentration decreased from 10 to about 5 mmol g-1 when the culture conditions changed from ammonia limitation to ammonia excess
B. Intracellular glutamate concentration increased from ~75 to 220 mmol g-1
C. Intracellular glutamine concentration increased linearly from about 4 mmol g-1 at 29 mM ammonia to about 27 mmol g-1 at 118 mM ammonia
D. With constant ammonia flux but increasing ammonia concentrations, the intracellular concentrations of glutamate and glutamine increase
VII. Northern (RNA) analyses
A. The purpose was to investigate whether RNA levels of nitrogen regulated genes changed with increasing ammonia concentrations
B. Amino acid permease-encoding genes versus biosynthetic genes
1. Amino acid permease-encoding genes were GAP1 and PUT4
2. The biosynthetic genes were ILV5 (α-acetoacetate reductoisomerase) and HIS4 (histidinol dehydrogenase).
C. Gene labelling
1. GDH1, GLN1 (glutamine synthetase [GS]), GAP1, ILV5, HIS4, ACT1 (actin), and H2A-H2B (histone) RNA levels were detected with 32P-labelled oligonucleotides
2. PUT4 RNA levels were analyzed with the oligonucleotide 5'-CTCCTCCTTCTTGGTGTCGCCGCCGCTACC-39
3. GDH2 RNA levels were detected with a 32P-labelled 2.5-kb XhoI-BamHI DNA fragment
D. The data were quantified with X-ray films at different exposure times
1. ACT1 RNA was used as the internal control for the amount of RNA blotted for the quantification of GAP1, PUT4, ILV5, HIS4, GDH1, and GDH2 RNA
2. H2A-H2B was used for the detection of GLN1 RNA levels
E. The expression levels of genes involved in the utilization of ammonia (GDH1, GDH2, and GLN1) were determined to study their responses to the amount of ammonia.
1. When ammonia concentrations increased up to 78 mM, the level of GDH1 RNA remained constant, but further increasing ammonia concentrations decreased the GDH1 RNA level
2. At ammonia concentrations of 29 and 44 mM no GDH2 RNA could be detected
3. When the ammonia concentration was increased to 61 mM the GDH2 RNA level increased
4. Maximum GLN1 expression occurred at 61 mM
5. Conclusion: the concentration of ammonia both repressed (GDH1) and induced (GDH2) the RNA expression of nitrogen-regulated genes
VIII. Effect of ammonia concentrations on GAP1 and PUT4
A. In previous studies it has been shown that
1. The expression of GAP1 is regulated in response to the ammonia concentration or the ammonia flux
2. The proline permease Put4p is regulated in a manner similar to that of Gap1p in response to ammonia
B. In this study, the influence of the ammonia concentration on GAP1 and PUT4 expression was analyzed at constant ammonia flux
1. At 29 and 44 mM ammonia the levels of GAP1 and PUT4 RNA were constant
2. From 44 mM ammonia upwards, the amounts of GAP1 and PUT4 RNA decreased
3. At 118 mM ammonia, almost no GAP1 RNA could be detected but PUT4 RNA was still present, although the amount was less than that at 44 mM ammonia
4. In an ammonia-limited continuous culture with varying levels of glucose:
a. With a small excess of glucose, a relation was observed between ammonia flux, residual ammonia concentration, and GAP1 expression
b. With a large excess of glucose, GAP1 expression did not change with increasing ammonia flux
C. Conclusion: GAP1 and PUT4 expression are regulated not by the ammonia flux but by the ammonia concentration.
D. An increased level of activity of the Gcn4p transcriptional regulator is caused by amino acid starvation which increases the transcription of biosynthetic genes
1. The RNA amount of the amino acid biosynthetic genes ILV5 and HIS4 increased with increasing extracellular ammonia concentrations (Maximal at about 66 mM)
2. As ammonia concentrations increased, the levels of ILV5 and HIS4 RNA decreased again
3. There was a similarity among the expression patterns of Gcn4p-regulated genes (GLN1, ILV5, and HIS4)
IX.Enzyme activities
A. Investigated if changed ammonia concentrations changed the levels of the activity of enzymes involved in the conversion of ammonia to glutamate or glutamine
1. Done by determining the levels of NADPH-glutamate dehydrogenase (GDH), NAD-GDH, and GS activity
a. NADPH-GDH and NAD-GDH activities were measured under Vmax conditions
b. GS activities were analyzed as described by Mitchell and Magasanik
2. Increasing ammonia concentration from 29 to 118 mM decreased the activity level of the NADPH-GDH from 4.1 to 1.8 μmol min-1 mg-1
3. Decreasing NADPH-GDH, decreased the level of GDH1 expression (showing the decrease is at least partly due to regulation on the transcriptional level)
4. The NAD-GDH activity level increased sharply (0.01 to 0.15 μmol min-1 mg-1) between 29 and 61 mM ammonia in the feed
a. further increase in the ammonia concentration did not result in a further increase in the level of NAD-GDH activity
B. The observed GDH2 RNA expression and NAD-GDH activity strongly suggested that this enzyme is regulated mainly at the level of transcription
C. Slight decreases in the levels of GS transferase activity (from 0.82 to 0.60 μmol min-1 mg-1) and GS activity (from 0.15 to 0.10 μmol min-1 mg-1) were observed with increasing ammonia concentrations up to 61 mM, but with further increasing ammonia concentrations no changes were observed
D. The altered level of GS activity was not caused by an altered transcription and therefore has to be posttranscriptional
E. The changes in NADPH-GDH and GS activity levels are reflected in the ammonia, a-ketoglutarate, glutamate, and glutamine concentrations.
X. Conclusion
A. The concentration of ammonia regulates the nitrogen metabolism of S. cerevisiae
1. The ammonia flux remained constant whereas the concentration of ammonia in the feed increased
2. Observed changes in nitrogen metabolism are regulated by either extracellular or intracellular concentrations of ammonia or by changes in levels of intracellular metabolites like a-ketoglutarate, glutamate, or glutamine
3. If the ammonia concentration is the regulator, S. cerevisiae may have an ammonia sensor, which could be a two-component sensing system for nitrogen
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