Sarah Carratt: Week 2

Instructions

1. Make a list of at least 10 biological terms for which you did not know the definitions when you first read the article. Define each of the terms. You can use the glossary in any molecular biology, cell biology, or genetics text book as a source for definitions, or you can use one of many available online biological dictionaries (links below). List the citation(s) for the dictionary(s) you use, providing a URL to the page is fine.
2. Write an outline of the article. The length should be the equivalent of 2 pages of standard 8 1/2 by 11 inch paper. Your outline can be in any form you choose, but you should utilize the wiki syntax of headers and either numbered or bulleted lists to create it. The text of the outline does not have to be complete sentences, but it should answer the questions listed below and have enough information so that others can follow it. However, your outline should be in YOUR OWN WORDS, not copied straight from the article.
   • What is the main result presented in this paper?
   • What is the importance or significance of this work?
   • What were the limitations in previous studies that led them to perform this work?
   • What were the methods used in the study?
   • Briefly state the result shown in each of the figures.
     • What do the X and Y axes represent?
     • How were the measurements made?
     • What trends are shown by the plots and what conclusions can you draw from the data?
   • What is the overall conclusion of the study and what are some future directions for research?
3. Each group of students will be assigned one section of the paper. The group will be responsible for explaining the section, including any tables/figures in detail to the class. Groups will be assigned on 1/20/11 in class. Dr. Dahlquist will prepare the PowerPoint slides this time; for future journal club assignments, you will prepare the PowerPoint.
   • Physiological parameters section, Figure 1: James, Nick
   • Northern analysis section, Figure 2: Carmen, Alondra
   • Enzyme activities section, Figure 3: Sarah

Online Sources

• Article: The Concentration of Ammonia Regulates Nitrogen Metabolism in Saccharomyces cerevisiae
• Biology Dictionary
• Gene Ontology
• NCI Dictionary of Cancer Terms

Student Response

Terms and Definitions

1. ammonia assimilation: The utilization of ammonia (or ammonium ions) in the net synthesis of nitrogen-containing molecules; e.g., glutamine synthetase. [1]
2. residual: Remaining or left behind. [2]
3. proline: One of the 20 amino acids directly coded for in proteins. [3]
4. biosynthetic: Relating to or produced by biosynthesis, which is the creation of more complex molecules from simpler molecules, such as the conversion of glucose to starch. [4]
5. gram-negative bacteria: Bacteria which lose crystal violet stain but are stained pink when treated by grams method. [5]
6. gram-positive bacteria: Bacteria that retain the stain or that are resistant to decolourisation by alcohol during grams method of staining. [6]
7. 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. [7]
8. biomass:The total mass of all living material in a specific area, habitat, or region. [8]
9. parameter: A variable whose measure is indicative of a quantity or function that cannot itself be precisely determined by direct methods, for example, blood pressure and pulse rate are parameters of cardiovascular function and the level of glucose in blood and urine is a parameter of carbohydrate metabolism. [9]
10. quantification:The expression of a numerical amount. [10]

Article Outline

Abstract

• "Saccharomyces cerevisiae" was grown in cultures with various amounts of ammonia.
• While the uptake of ammonia proved to be constant, there was a difference in net output when the culture was given more ammonia.
• Increases in ammonia resulted in:
  • Higher concentrations of intracellular glutamate and glutamine
  • Increases in levels of NAD-dependent glutamate dehydrogenase activity and its mRNA (gene GDH2)
  • Decreases in levels of NADPH-dependent glutamate dehydrogenase activity and its mRNA (gene GDH1)
  • Decreases in the levels of mRNA for the amino acid permease-encoding genes GAP1 and PUT4
• Nitrogen metabolism is likely to be based on the abundance of ammonia and the ability to consume the ammonia longer because of its excess.
• Nitrogen metabolism is NOT likely to be based on a change in the rate of consumption because of excess ammonia.

Introduction

• Background:
  • "Saccharomyces cerevisia" get nitrogen from ammonia.
  • Ammonia allows for better growth when compared to proline or urea.
• Other Research:
  • Ammonia concentration is the thing to focus on.
• Important variables to consider:
  • External ammonia concentration
  • Rate of ammonia assimilation (i.e., the ammonia flux)

Materials and Methods

• Yeast was grown in cultures with:
  • Ammonia concentrations of 29, 44, 61, 66, 78, 90, 96, 114, and 118 mM
  • Glucose concentration of 100 mM
  • Dilution rate of 0.15 h21
• Measurements were taken of ammonia concentrations and biomass.
• Calculated ammonia flux:
  • dilution rate x (input ammonia concentration - residual ammonia concentration)/biomass
• Analysis:
  • Northern (RNA) analyses
    • Did nitrogen-regulated genes change with increasing ammonia concentrations?
  • Mitchell and Magasanik methods:
    • NADPH-GDH and NAD-GDH measured under Vmax conditions
    • GS activities were analyzed

Results

• The Numbers!
  • Increase ammonia from 29 to 61 mM = increase of the biomass from 4.9 to 8.2 g/liter with “‘constant”’ leftover ammonia concentration in the culture (0.022 mM)
  • Higher than 61 mM, the residual ammonia concentration increased at a constant rate to 62 mM and the biomass remained at about 8.2 g/liter while glucose (food source) became limiting.
  • The ammonia flux into biomass was about 1.1 mmol/[(h)(g)]

Discussion

• Concentration of ammonia controls the nitrogen metabolism of “S. cerevisiae”
• Results are known because:
  • Ammonia consumption rate remained constant
  • Concentration of ammonia in the feed increased
• Possibilities for regulation:
  • Extracellular or intracellular concentrations of ammonia
  • Changes in levels of intracellular metabolites like a-ketoglutarate, glutamate, or glutamine.
• Implications:
  • If the ammonia concentration is the regulator…
    • “S. cerevisiae” might have an ammonia sensor which could be a two-component sensing system for nitrogen
    • “S. cerevisiae” might be similar to gram-negative bacteria

References

1. Boles, E., W. Lehnert, and K. Zimmermann. 1993. The role of the NADdependent glutamate dehydrogenase in restoring growth of a “Saccharomyces cerevisiae” phosphoglucose isomerase mutant. Eur. J. Biochem. 217:469–477.
2. Courchesne, W. E., and B. Magasanik. 1983. Ammonia regulation of amino acid permeases in Saccharomyces cerevisiae. Mol. Cell. Biol. 3:672–683.
3. Dever, T. E., L. Feng, R. C. Weh, A. M. Cigan, T. F. Donahue, and A. G. Hinnebusch. 1992. Phosphorylation of initiation factor 2a by protein GCN2 mediates gene specific translational control of GCN4 in yeast. Cell 68:585–596.
4. Magasanik, B. 1988. Reversible phosphorylation of an enhancer binding protein regulates the transcription of bacterial nitrogen utilization genes. Trends Biochem. Sci. 13:475–479.
5. Magasanik, B. 1992. Regulation of nitrogen utilization, p. 283. In J. R. Broach, E. W. Jones, and J. R. Pringle (ed.), The molecular and cellular biology of the yeast Saccharomyces: gene expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
6. Miller, S. M., and B. Magasanik. 1991. Role of the complex upstream region of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae. Mol. Cell. Biol. 11:6229–6247.
7. Mitchell, A. P., and B. Magasanik. 1983. Purification and properties of glutamine synthetase from Saccharomyces cerevisiae. J. Biol. Chem. 258:119–124.
8. Mitchell, A. P., and B. Magasanik. 1984. Three regulatory systems control production of glutamine synthetase. Mol. Cell. Biol. 4:2767–2773.
9. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: A laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
10. Sierkstra, L. N., E. G. ter Schure, J. M. A. Verbakel, and C. T. Verrips. 1994. A nitrogen-limited, glucose repressed, continuous culture of “Saccharomyces cerevisiae.” Microbiology 140:593–599.
11. Sierkstra, L. N., J. M. A. Verbakel, and C. T. Verrips. 1992. Analysis of transcription and translation of glycolytic enzymes in glucose-limited continuous cultures of Saccharomyces cerevisiae. J. Gen. Microbiol. 138:2559–2566.
12. ter Schure, E. G., H. H. W. Sillje´, L. J. R. M. Raeven, J. Boonstra, A. J. Verkleij, and C. T. Verrips. 1995. Nitrogen-regulated transcription and enzyme activities in continuous cultures of Saccharomyces cerevisiae. Microbiology 141:1101–1108.
13. Wiame, J.-M., M. Grenson, and H. N. Arst, Jr. 1985. Nitrogen catabolite repression in yeast and filamentous fungi. Adv. Microb. Physiol. 26:1–88

Questions

1. In this experiment, the scientists grew budding yeast in ammonia and measured the net differences that resulted from varying this amount of ammonia. The results were that nitrogen metabolism is correlated with ammonia abundance and increases in nitrogen can likely to be attributed to the yeast's ability to infinitely consume ammonia at a constant rate. Nitrogen metabolism is NOT likely to be based on a change in the rate of consumption because of excess ammonia.
2. Because yeast is a model organism, the significance of this work lies in the understanding of other living things and our ability to manipulate genes and environmental conditions to get the results we desire.
3. In previous studies, the external ammonia and ammonia assimilation were combined into one variable and the possibility of a flux was not able to be tested.
4. They used the northern (RNA) analyses and methods outlined by Mitchell and Magasanik.
5. Results in the figures:
   1. Figure 1:
      1. X:
      2. Y:
      3. Measurements:
      4. Trends:
   2. Figure 2:
      1. X:
      2. Y:
      3. Measurements:
      4. Trends:
   3. Figure 3:
      1. X:
      2. Y:
      3. Measurements:
      4. Trends:
6. Overall, nitrogen metabolism is not likely to be based on an increased assimilation of ammonia, but on the general concentration of ammonia that is present.

Links

• BIOL398-01/S11:Assignments
• BIOL398-01/S11:People
• BIOL398-01/S11:Sarah Carratt