Moneil5 Week 3

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Margaret J. ONeil

Assignment Pages:

Personal Journal Entries:

Shared Journal Entries:

Purpose

The purpose of this assignment is to prepare for the first journal club, and to learn more about the impact of limiting agents, such as nitrogen, on the metabolism of Saccharomyces cerevisiae.

Definitions

  • α-ketoglutarate: a compound that has important roles in carbohydrate and amino‐acid metabolism, especially in transamination reactions and as a component of the TCA cycle. (Oxford Reference, 2017)
  • glutarate: the dianion of glutaric acid; any mixture of free glutaric acid and its mono- and dianions (Oxford Reference, 2017)
  • glutamine: An amino acid commonly found in proteins. Crosslinks between glutamine and lysine side groups are important in stabilizing the structure of protein aggregates such as fibrin. Tissue culture media for animal cells are commonly enriched tenfold for glutamine, acting as a carbon source. (Oxford Reference, 2017)
  • oligonucleotides: a short polymer of nucleotides
  • proline: a heterocyclic, non-polar imino acid that is present in all proteins observed thus far. 9Oxford Reference, 2017)
  • permease:A membrane-bound protein in bacteria that is responsible for transport of a specific substance in or out of the cell; sometimes referred to as a transport protein. (Oxford Reference, 2017)
  • dehydrogenase: any enzyme that catalyses the removal of hydrogen atoms in biological reactions. Dehydrogenases occur in many biochemical pathways but are particularly important in driving the electron-transport-chain reactions of cell respiration. They work in conjunction with the hydrogen-accepting coenzymes NAD and FAD. (Oxford Reference, 2017)
  • biosynthetic: of or relating to biosynthesis; any substance produced by biosynthesis. (Oxford Reference, 2017)
  • metabolites: a chemical compound that is produced or consumed during metabolism; include low molecular weight compounds that are produced or converted by enzymes during metabolism or the precursors or breakdown products of biopolymers. (Oxford Reference, 2017)

Outline of the ter Schure (1995) Paper

Main Results of Paper

  • The concentration of ammonia regulates nitrogen metabolism in Saccharomyces cerevisiae
    • Determined to be regulated either through extracellular or intracellular concentrations, or through changes in levels of metabolites such as α-ketoglutarate, glutamine or glutarate
    • Ammonia concentration as a regulator implies budding yeast has an ammonia sensor, such as that found in gram-negative bacteria. A two-component sensing system for nitrogen

Significance of Paper

  • This paper shows the regulatory nature of ammonia concentration on the nitrogen metabolism of S. cerevisiae, and through knowing that the concentration of ammonia rather than flux has an impact on the metabolism of budding yeast, we can learn more about how the various metabolic pathways for yeast interact with each other.
    • Understanding the relationship of ammonia concentration to nitrogen metabolism can also be helpful in a commercial setting, such as cultivating large quantities of yeast for baking, brewing beer, or producing yeast extract
  • Understanding the genes that play a role in nitrogen metabolism in S. cerevisiae can reveal how the gene regulatory networks and metabolic pathways are connected, which might tell us more about how our own GRNs and metabolic pathways are connected

Limitations of Previous Work

  • Previous studies similarly looked into the role of ammonia concentration on being a key influencer for the regulation of nitrogen metabolism. These studies didn't control ammonia flux however, so it is possible that ammonia flux rather than concentration, or flux combined with concentration are the key influencers on regulation rather than just ammonia concentration. In their study, the authors of this paper are discriminating between continuous cultures having the same levels of flux but having different concentrations of ammonia

Methods Used in this Study

The authors of this paper used the following methods to conduct their study:

Physiological parameters

  • S. cerevisiae SU32 was grown in 9 different continuous cultures with differing concentrations of ammonia but the same concentration of glucose (100mM) in the feed medium at a dilution rate of 0.15 1/h.
    • Ammonia concentrations were 29, 44, 61, 66, 78, 90, 96, 114, and 118 mM
  • Ammonia and biomass concentrations were then calculated, and from these values and the residual ammonia concentration, the ammonia flux was calculated and found to be the same value across concentrations.

Northern Analyses

  • Northern analyses (using RNA) was used to determine if known nitrogen-regulating genes experienced/exhibited changing RNA levels with the increasing concentrations of ammonia
    • The amino acid permease-encoding genes were GAP1, PUT4 with the biosynthetic genes ILV5 and HIS4.
  • RNA levels for the genes GDH1, GLN1, GAP1, ILV5, HIS4, ACT1, and H2A-H2B were detected using P=labelled oligonucleotides
  • RNA levels for PUT4 were analyzed using a different specific nucleotide
  • RNA levels for GDH2 were detected using a P-labelled 2,5-kb Xho1-BAMHI DNA fragment
  • The data for the above genes was quantified using X-Ray film with different exposures
    • In quantifying GAP1, PUT4, ILV5, HIS4, GDH1, and GDH2, ACT1 RNA was used as the control for the interior of the cell and amount of RNA blotted.
    • In detecting and quantifying GLN1 RNA levels, H2A-H2B was used
  • In order to study their responses to the amount of ammonia present, expression levels of genes GDH1, GDH2, and GLN1 were determined (all involved in the utilization of ammonia)
  • Based on previous studies done on PUT4 and GAP1, the influence of ammonia concentration on the expression of those genes was investigated at a constant flux as these two genes were previously seen to be regulated in regards to ammonia concentration or ammonia flux.
  • GAP1 was placed in an ammonia -limited culture with a large excess of glucose to see if GAP1 expression changed with increasing ammonia flux

Enzyme Activities

  • To look into whether changing ammonia concentrations resulted in changes in the levels of enzyme activity in enzymes that were involved in the conversion of ammonia into glutamate/glutamine, NADPH-glutamate dehydrogenase, NAD-GDH and GS activity was determined
    • Activities of NADPH-GDH and NAD-GDH were measured under Vmax conditions, and GS activities were analyzed as described in a paper done by Mitchell and Magasanick

Analysis of Figures

Figure 1

1A
  • The X-axis for this figure shows ammonia concentration in mM, and the left Y-axis shows residual ammonia concentration in mM ( open squares connected by lines) while the Y-axis on the right shows the biomass in dry grams as gl-1 (open triangles) and the furthest right Y-axis measures ammonia flux in terms of mmolg-1 h-1 (filled in circles)
  • These measurements were made through growing S. cerevisiae in continuous culture as described in the physical parameters portion of the methods section
  • This figure shows that where the ammonia concentration increased from 29-61 mM, there was an increase in the biomass which demonstrates ammonia limitation. There is a point where biomass stays the same, at an ammonia concentration of around 61mM, which implies that at this point glucose becomes the limiting reagent for nitrogen metabolism. The ammonia flux was calculated as well, and is shown to be relatively constant across all ammonia concentrations
1B
  • The X-axis for this figure shows ammonia concentration in mM. The furthest left Y-axis shows oxygen consumption in terms of mmolg-1 h-1 (shaded triangles), the second Y-axis shows carbon dioxide production in mmolg-1 h-1 (shaded squares) and the 3rd Y-axis shows the respiratory quotient, which has no units (open circles).
  • The measurements in this figure were made through monitoring the carbon metabolism of the yeast in terms of oxygen consumption and carbon dioxide production as the ammonia concentration increased.
  • When the ammonia concentration was above 44mM, the respiratory quotient was found to be relatively constant, and therefore so was the oxygen consumption and carbon dioxide production.
    • When ammonia was limiting, however, the oxygen consumption decreased and the carbon dioxide production increased, while the residual glucose concentration stayed the same
    • Above findings indicate except when ammonia is limiting, no significant changes in carbon metabolism was seen as the concentration of ammonia increased
1C

Figure 2

Figure 3

Conclusion of Study

Future Directions of Research

Acknowledgements

  • Discussed who would be presenting which part of each figure over text several times with Lauren Kelly, and Cameron M. Rehmani Seraji
  • Researched all definitions through Oxford Reference (see references section below for full citations)
  • Used a printed copy of the ter Schure article for all analysis of paper
  • Except for what is noted above, this individual journal entry was completed by me and not copied from another source

Margaret J. Oneil 00:01, 2 February 2017 (EST)

References

Α-ketoglutarate - Oxford Reference. (2017, January 15). Retrieved February 01, 2017, from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803095405413

Biosynthetic - Oxford Reference. (2017, January 15). Retrieved February 01, 2017, from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803095507499

Dahlquist, Kam D. (2017) BIOL398-05/S17:Week 3. Retrieved from http://www.openwetware.org/wiki/BIOL398-05/S17:Week_3 on 1 February 2017.

Dehydrogenase - Oxford Reference. (2017, January 15). Retrieved February 01, 2017, from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803095707657

Glutamine - Oxford Reference. (2017, January 15). Retrieved February 01, 2017, from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803095856227

Glutarate - Oxford Reference. (2017, January 15). Retrieved February 01, 2017, from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803095856253

Metabolite - Oxford Reference. (2017, January 15). Retrieved February 01, 2017, from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803100152732

Oligonucleotide - Oxford Reference. (2017, January 15). Retrieved February 01, 2017, from http://www.oxfordreference.com/view/10.1093/oi/authority.20110810105523386

Permease - Oxford Reference. (2017, January 15). Retrieved February 01, 2017, from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803100318301

Proline - Oxford Reference. (2017, January 15). Retrieved February 01, 2017, from http://www.oxfordreference.com/view/10.1093/oi/authority.20110803100349185

ter Schure, E. G., Sillje, H. H., Verkleij, A. J., Boonstra, J., & Verrips, C. T. (1995). The concentration of ammonia regulates nitrogen metabolism in Saccharomyces cerevisiae. Journal of bacteriology, 177(22), 6672-6675