Elizabeth Polidan Week3

Elizabeth Polidan

BIOL 398.03 / MATH 388

• Loyola Marymount University

• Los Angeles, CA, USA

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Week 3 Assignment

Vocabulary

1. Permease
   1. (Science: enzyme) 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. Reference: http://www.biology-online.org/dictionary/Permease
2. Allosteric inhibition
   1. See allosteric Enzyme. Inhibitors act as 'modulators' in enzyme execution as they can attach themselves to a molecule that will alter the binding Site for the enzyme, rendering it unusable and therefore rendering the enzyme inactive. Reference: http://www.biology-online.org/dictionary/Allosteric_Inhibitors
3. Northern analysis
   1. A procedure similar to the southern blot analysis, 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. Reference: http://www.biology-online.org/dictionary/Northern_blot_analysis
4. Oligonucleotide
   1. A linear sequence of up to 20 nucleotides joined by phosphodiester bonds. Above this length the term polynucleotide begins to be used. A short sequence of [[nucleotides. Reference: http://www.biology-online.org/dictionary/Oligonucleotide
5. GLN1
   1. Glutamine synthetase (GS); synthesizes glutamine from glutamate and ammonia; with Glt1p, forms the secondary pathway for glutamate biosynthesis from ammonia; expression regulated by nitrogen source and by amino acid limitation; relocalizes from nucleus to cytoplasmic foci upon DNA replication stress. Reference: http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=GLN1
6. H1S4
   1. Enocdes histidinol dehydrogenase Reference: ter Schure, E.G., H.H.W. Silije, L.J.R.M. Raeven, J. Boonstra, A.J. Verkleij, and C.T. Verrips, 1995, Nitrogen-regulated transcription and enzyme activities in continous cultures of Saccharomyces cerevisiae, Microbiology, 141:1101-1108.
7. ILV5
   1. Bifunctional acetohydroxyacid reductoisomerase and mtDNA binding protein; involved in branched-chain amino acid biosynthesis and maintenance of wild-type mitochondrial DNA; found in mitochondrial nucleoids (1, 2, 3, 4) Reference: http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=ILV5

Article Outline

• Introduction
  • Studies have shown that ammonia is the best source of nitrogen for Saccharomyces cerevisiae.
  • Previous studies have indicated ammonia concentration is key in nitrogen metabolism. However those studies have allowed ammonia concentration and ammonia flux to both vary. This study was designed to hold the flux constant and allow concentration to vary. It looked at gene expression and enzyme activity to explore nitrogen regulation.
  • This information will help understand the regulation of cell growth.
• Methods
  • Saccharomyces cerevisiae was grown in cultures with continuous feed
    • Incoming concentrations of ammonia were varied from a low of 29 milimoles per liter (mM) to a high of 118 mM.
    • Incoming concentrations of glucose were all held at 100 mM
    • The dilution rate was 0.15/hour
  • Physiological parameters were monitor to determine the state of the yeast in the system
    • Biomass was measured in grams/liter
    • Residual ammonia was measured mM
    • Ammonia flux was calculated from resulting biomass, input ammonia concentration and residual ammonia concentration: NH4 flux = (diluation rate x (incoming ammonia concentration – residual ammonia concentration)/biomass)
    • Oxygen consumption was measured in mM per gram biomass per hour (mM/g/h)
    • Carbon Dioxide production was measured in mM/g/h
    • Respiratory quotient was calculated as (CO2 produced/O2 consumed)
    • Measured alpha-ketoglutarate at varying ammonia concentration
    • Measured glutamate at varying ammonia concentration
    • Measured glutamine at varying ammonia concentration
  • Northern analysis
    • Measured RNA to determine if levels varied with ammonia concentration in nitrogen regulated genes
      • GDH1, GDH2
      • GAP1, PUT4 -
      • ILV5, HIS4, GLN1 - Gen4p-regulated genes
  • Enzyme activities
    • Levels of NADPH-glutamate dehydrogenase, NAD-GDH, and GS activity were measured to determine if there was any change in enzyme activity as the ammonia concentration was varied.
• Discussion
  • Figure 1 - The physiological parameters of the experiment
  • Frame A
    • x-axis is ammonia concentration in mM
    • y-axis (left side of plot) is residual ammonia concentration in mM which held steady at 0.022 mM until inflow ammonia concentration increased beyond 61 mM. Then the residual levels rose linearly with increase in ammonia levels. In other words, the ammonia was not being assimilated beyond concentrations of 61 mM.
    • y-axis (right side of plot) is biomass in grams per liter. It shows an increase in biomass as the ammonia concentration increases, up to 61 mM. For concentrations beyond 61 mM there was no increase in biomass. This indicates that glucose had become the metabolic limiter.
    • y-axis (far right side of plot) is ammonia flux. This level remained relatively constant as per experimental design.
    • Frame B
      • x-axis is ammonia concentration in mM.
      • y-axis (left side) is O2 consumption and CO2 production in mM/g/l.
      • y-axis (right side) is respiratory quotient (CO2 produced divided by O2 consumed).
      • When the ammonia levels were limited (<= 44 mM) there were differences in CO2 production and O2 consumption. Once ammonia was not a limiting factor and became excessive levels of CO2 production and O2 consumed and the respiratory quotient remained constant.
    • Frame C
      • x-axis is ammonia concentration in mM for all three plots in Frame C.
      • y-axis (plot 1) is α-ketoglutarate concent in μMol/g.
        • Once ammonia reached some threshold concentration (between 0.44 and 0.61 mM) the level of α-ketoglutarate decreased as it reacted with the ammonia to form glutamate. As the level of ammonia became excessive the α-ketoglutarate leveled off and remained constant.
      • y-axis (plot 2) is glutamate concentration in μMol/g.
        • When ammonia went from limited to excess the glutamate level increased.
      • y-axis (plot 3) is glutamine concentration in μMol/g.
      • The glutamine concentration increased linealry with the increase in ammonia concentration.
  • Figure 2 - RNA levels from Nitrogen-Regulated Genes
    • Left Frame
      • x-axis is ammonia concentration in mM
      • y-axis GDH1 and GDH2 intensity ratios relative to the control RNA ACT1. In the GDH1 plot, the ratio at an ammonia concentration of 29 mM was set to 100%, then all others were scaled relative to that point. In the GDH2 plot, the ratio at an ammonia concentration of 118 mM was set to 100%, then all others were scaled relative to that point.
    • Middle Frame
      • x-axis is ammonia concentration in mM
      • y-axis is GAP1 and PUT4 intensity ratios relative to the control RNA ACT1. The ratios at an ammonia concentration of 29 mM was set to 100%, then all others were scaled relative to that point.
    • Right Frame
      • x-axis is ammonia concentration in mM
      • y-axis is GLN1, H1S4, and ILV5 intensity ratios relative to the control RNA ACT1. The ratios at an ammonia concentration of 29 mM was set to 100%, then all others were scaled relative to that point.
        • The levels of GLN1, H1S4 and ILV5 all increased with increased concentration of ammonia, peaking when ammonia was at 66 mM. The curves of the three genes were quite similar.
  • 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?
• Conclusion
  • It is the ammonia concentration that regulates nitrogen metabolism in S. cerevisiae, not the flux level.
    • The driver of the observed changes was either ammonia concentration itself (intra- and intercellular) or ammonia concentration-driven changes in levels of metabolites such as glutamate or glutamine.