Alondra Vega: Week 2

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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

Student Response

Terms and Definitions

  • ammonia:a strongly basic, irritating, colorless gas, which is lighter than air and readily soluble in water. It is often formed in nature as a by-product of protein metabolism in animals. [1]
  • ammonia assimilation:The utilization of ammonia (or ammonium ions) in the net synthesis of nitrogen-containing molecules. An example is glutamine synthetase, which will be mentioned in the Schure et al. paper. [2]
  • flux:he total amount of a quantity passing through a given surface per unit time. [3]
  • parameter:A variable whose measure is indicative of a quantity or function that cannot itself be precisely determined by direct methods. [4]
  • biomass:The total mass of all living material in a specific area, habitat, or region. [5]
  • residual:Remaining or left behind. [6]
  • concentration:The ratio of the mass or volume of a substance (solute) to the mass or volume of the solvent or solution.[7]
  • dehydrogensae:enzyme that oxidizes a substrate by transferring hydrogen to an acceptor that is either NAD/NADP or a flavin enzyme. [8]
  • metabolism:The process involving a set of chemical reactions that modifies a molecule into another for storage, or for immediate use in another reaction or as a by product. [9]
  • biosynthetic:Relating to or produced by biosynthesis. Biosynthesis is the building up of a chemical compound in the physiologic processes of a living organism.[10] [11]

Template:Alondra Vega

Outline: The Concentration of Ammonia Regulates Nitrogen Metabolism in Saccharomyces cerevisiae

Abstract

  • Sacchoromyces cerevisiae (S. cerevisiae) was grown in a continuous culture with some input of ammonia concentrations.
  • The rate of ammonia assimilation proved to be constant.
    • These cultures were used to see the effects of nitrogen limitation to nitrogen excess and glucose limitation.
  • They found that by increasing the ammonia concentrations outside the cell, the glutamate and glutamine inside the cell also increased.
  • The increases in ammonia also correspond to the increases in NAD-dependent glutamate dehydrogenase activity, decreases in dehydrogenase activity and decreases in the levels of mRNA.
  • From this paper, it may be seen that the main factor of nitrogen metabolism might be concentration of ammonia not how much of it passes through the cell.

Introduction

  • S. cerevisiae
    • They prefer ammonia as their primary nitrogen source.
    • Compared to proline and urea, ammonia will allow for better growth.
    • The components of nitrogen metabolism can be regulated at the level of gene expression and of enzyme activity.
  • Previous research has shown the importance of ammonia concentrations.
  • Since the cultures differ in external ammonia concentration and in the rate of ammonia assimilation, then it was believed that the rate was the parameter which was in control, not the concentration.
  • In this research paper the focus is ammonia concentration, thus some cultures may have had the same flux.
  • The ammonia concentration is the parameter for this experiment.

Materials and Methods

  • Strain used: S. cerevisiae SU32
    • It was grown in continuous cultures that contained different amounts of ammonia concentrations.
      • The concentrations were 29, 44, 61, 66, 78, 90, 96, 114, and 118 mM.
      • They also had a fixed glucose concentration of 100 mM.
    • The dilution rate that was used was 0.15 h-1.
  • Both ammonia and biomass were measured.
  • The ammonia flux was measured by the dilution rate × (input ammonia concentration – residual ammonia concentration)/biomass.
  • There were two types of analyses that were done.
  1. Northern Analysis: was used to see whether the RNA levels of nitrogen regulated genes would change with the increase ammonia concentrations.
      • RNA levels were measured with a 32P-labelled oligonucleotides. Note: Each gene had its own specific label.
  1. Mitchell and Magasanik: were used to analyze the glutamine synthetase (GS) activity. They were measured under Vmax conditions.

Results

  • The increase in the ammonia concentrations was from 29 to 61 mM, which also resulted in an increase of the biomass from 4.9 to 8.2 g liter-1.
    • The residual was constant at about 0.022 mM.
  • When the ammonia concentrations increased higher than 61 mM, the residual ammonia concentration also increased linearly to 62 mM.
  • The ammonia flux into the biomass was 1.1 mmol 1/(g×h).
  • The CO2 production and O2 consumption remained relatively constant when the ammonia concentration was above 44 mM.
  • Figure 1B (which will be discussed later) showed that there was no significant changes in the carbon metabolism when ammonia was increased. The only change was when the concentration was 29 mM.
  • The intracellular glutamine concentrations increased linearly from 4μmol/g at 29 mM of ammonia to 27 μmol/g at 118 mM.
  • When ammonia levels increased to 61 mM, there was also an increase in the GDH2 RNA level.
  • They also found that from 44 mM ammonia upwards, the amounts of GAP1 and PUT4 RNA decreased, while it stayed constant at 29 mM and 44 mM.
  • When the ammonia concentrations increased from 29 to 118 mM, the activity levels of NADPH-GDH decreased from 4.1 to 1.8.

Figures

  • Figure 1
    • A: Shows the ammonia and biomass concentrations. The x-axis shows the ammonia concentrations while the y-axis shows both the residual ammonia concentration along with the biomass. It also shows the ammonia flux which is kept relatively constant. One can see from the figure that the residual stays constant after 60 mM, while the biomass keeps increasing when the ammonia concentration increases.
    • B:In the x-axis the ammonia concentration is displayed, while in the y-axes both the O2 consumption and CO2 production, and the respiratory quotient is displayed. It is clear that as ammonia concentration increases, O2 consumption also increases and then stays constant after 40 mM. Also, both the respiratory quotient and the CO2 production decrease. The respiratory quotient settles at about 42 mM and then stays constant, while the CO2 production settles at about 60 mM and then stays constant.
    • C: In the x-axis the ammonia concentrations are shown, while the y-axes show the levels of α-ketogluterate, glutamate and glutamine. As ammonia concentrations increase, α-ketoglutarate decreases, while both glutamate and glutamine increases.
  • Figure 2
    • In the x-axis it shows the ammonia concentrations, while in the y-axis it shows the percent expression of nitrogen regulated genes found in RNA. As ammonia concentrations increases, GDH1, GAP1 and PUT4 decrease in their expression. As ammonia concentration increases, GDH2, HIS4, ILV5 and GLN1 increase. As the concentrations get really high, eg. after 100 mM, some of the genes whose expression increased, decreases for the most part.
  • Figure 3
    • In the x-axis the ammonia concentrations are shown, and the y-axis shows the GS transferase, NAD-GDH, and NADPH-GDH levels. As ammonia concentrations, the NADPH-GDH decreases and NAD-GDH increases. As ammonia increases, GS transferase is constant after 60 mM, but it does start off high.

Discussion

  • The glutamine and glutamate inside the cell increases, as ammonia concentrations increase and the ammonia flux stays constant.
  • The concentration of ammonia stops the expression of GDH1 and increases the production of GDH2, which are nitrogen-related genes.
  • When ammonia is limited in a culture and there is a lot of glucose, the expression of GAP1 did not change.
  • GAP1 and PUT4 are regulated by ammonia concentration, not by the ammonia flux.
  • The decrease in NADPH-GDH was followed by a decrease in the level of expression in the level of GDH1, thus the decrease is partly due to regulation on the transcriptional level.
  • The RNA expression of GDH2 along with NAD-GDH suggested that this enzyme is regulated mainly at the level of transcription.
  • This paper demonstrated that the concentration of ammonia regulates the nitrogen metabolism of yeast in many levels.
  • The nitrogen-metabolism is either regulated by inside or outside ammonia concentration in the cell or by the changes in the metabolites.
  • There is an implication that S. cerevisiae has an ammonia sensor, which can be shown to be a two-component sensing system fro nitrogen. These types of two-component systems have been found in 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
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