Nick Rohacz: Week 2

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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.

  1. Dilution rate - A solution that is diluted per unit of time, in this case .15/hr.[1]
  2. Ammonia Limitations - That which limits; a restriction; qualification; a restraining condition. [2]
  3. Ammonia Flux - Total amount of ammonia passing through a given surface over a unit of time. [3]
  4. Oligonucleotide - An oligonucleotide" is a short nucleotide sequence, generally made up of less than 20 nucleotides. [4]
  5. Amino acid starvation - Lengthy and continuous deprivation of Amino Acids. [5]
  6. Two-component sensing system - A regulatory system that controls gene expression in response to nitrogen-source availability. [6]
  7. Gram-negative bacteria - Bacteria which lose crystal violet stain but are stained pink when treated by grams method. [7]
  8. Proline - One of the 20 amino acids directly coded for in proteins. [8]
  9. Biomass - The total mass of all living material in a specific area, habitat, or region. [9]
  10. Northern RNA analysis- An electroblotting method in which rNA is transferred to a filter and detected by hybridisation to (32)P labelled RNA or dNA. [10]

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.
         o What do the X and Y axes represent?
         o How were the measurements made?
         o 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? 

Contents

Article Outline

Abstract

Saccharomyces cerevisiae

  • Varying concentrations in cultures.
  • Constant dilution rate
  • Fixed glucose conscentration
  • Seems to depend on Ammonia concentration rather than Ammonia Flux.

Introduction

Known

  • Ammonia preferred nitrogen supply source for Sacchomyces cerevisiae instead of Proline or Urea.
    • Growth occurs at a faster rate
    • Allows for regulation of genes and enzyme activity.
    • Ammonia concentration more important than ammonia assimilation
      • Ammonia concentration varies
      • Ammonia flux and glucose concentrations constant.

Materials and Methods

  • Saccharomyces cerevisiae SU32
    • Physiological Parameters
      • Ammonia Concentration 29,44,61,66,78,90,96,114,118 mM
      • Ammonia Flux of 1.1 mmol g^-1 hr^-1
      • Fixed glucose concentration of 100 mM
      • Dilution Rate 0.15 hr^-1
  • Measurements were taken of biomass and ammonia concentrations as Ammonia is used up
    • Northern Analysis
      • Amino acid permease encoding genes, GAP1 PUT4
      • Biosynthetic genes ILV5, HIS4, GDH1, GLN1, GS, ACT1,H2A-H2B
  • Expression levels were watched for varying RNA's, used x-ray film with different exposure times
    • Enzyme Activities
      • Activity levels of NADPH-GDH, NAD-GDH, GS transferase, GS synthetase
  • Determined levels of enzymes at varying concentrations.

Results

  • Physiological Parameters
    • For 29-61 mM, biomass increased from 4.9 to 8.2 g liter^-1, residual concentration was .022 mM
    • For 62-118 mM, biomass remained at 8.2 g liter^-1,
    • CO2 and O2 consumption constant for all concentrations above 44 mM, 29-44 mM had varying consumption rates, see 1B
    • Ammonia Flux stayed constant, leading to an increase in glutamate and glutamine concentrations.
  • Northern Analysis
    • GDH1; 29-78 mM, RNA remains constant; >78 mM, RNA decreased
    • GDH2; 29-44 mM, No RNA; >61 mM, increase in RNA
    • GLN1; Max expression occured at 61 mM
    • GAP1; 29-44 mM, RNA constant; >44 mM, concentrations decreased
  • Enzyme Activites
    • NADPH-GDH, 29-118 mM, activity decreased from 4.1 to 1.8
    • NADH-GDH, 29-61 mM, activity increased from .01 to .15; >61 mM results in no change
    • GS, 29-61 mM, decrease in GS transferase and GS activity, .82 to .60 and .15 to .10, respectively.

Discussion

  • Based on all of the data, it is evident that the nitrogen metabolism, specifically enzyme activity and gene expression, is dependent on both the intracellular and extracellular ammonia concentrations, rather than the ammonia flux.


Questions

  • The main result in this paper is that no matter the Ammonia Flux, the activity and expression of enzymes all depends on the concentration of ammonia.
  • The significance of this work is that yeast has a nitrogen metabolism like many organisms, and a very little amount of genes, this allows for easy testing for our own metabolisms.
  • The external ammonia and ammonia assimilation were combined into one variable and the possibility of a flux was not able to be tested.
  • Northern RNA analysis.
  • ====Figure 1====
    • 1A - Residual Ammonia(y-axis 1) increases for all concentrations, Ammonia Flux(y-axis 2) is constant for all concentrations, Biomass (y-axis 3) increases up to 61 mM, constant after that; all this happens for varying, increasing ammonia concentrations(x-axis)
    • 1B - O2 consumption(y-axis 1) increases drastically for 29-44 mM, constant for all other concentrations; CO2 production(y-axis 2) decreases drastically for 29-44 mM, constant for all other concentrations; Respiratory Quotient(y-axis 3) decreases drastically from 29-44 mM and relatively constant for other concentrations; All this happens as Ammonia Concentration increases(x-axis)
    • 1C - ketoglutarate(y-axis) decreases, while glutamate(y-axis) and glutamine(y-axis) increase for all ammonia concentrations(x-axis)
  • ====Figure 2====
    • % Expression vs Ammonia Concentration, first graph consists of % expression of GDH2 and GDH1, second graph consists of % expression of GAP1 and PUT4, third graph consists of GLN1, HIS4 and ILV5
  • ====Figure 3====
    • NADPH-GDH(y-axis 1) vs ammonia concentration, NADH-GDH(y-axis 2) vs ammonia concentration, GS transferase(y-axis 3) and GS synthetase(y-axis 4) vs ammonia concentraion
  • The overall conclusion is that Nitrogen Metabolisms are not controlled by ammonia flux but instead by ammonia concentration.|
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