Kasey E. O'Connor Week 3 Journal

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(fix link)
(northern analyses)
Line 33: Line 33:
**the intracellular glutamate concentration (y-axis) increased from about 75 to 220 and the glutamine concentration (y-axis) increased from approximately 4 to about 27
**the intracellular glutamate concentration (y-axis) increased from about 75 to 220 and the glutamine concentration (y-axis) increased from approximately 4 to about 27
====Northern Analyses====
====Northern Analyses====
-
*
+
*Northern Analyses were preformed see whether the RNA levels of nitrogen regulated genes changed with increasing ammonia concentrations
-
*
+
*after being analyzed, the RNA data was quantified with X-ray films at different exposure times
-
*
+
*Figure 2 showed that when the ammonia concentrations (x-axis) were increased up to 78 mM, the level of GDH1 RNA (y-axis) remained constant, but when it kept being increased the GDH1 RNA level decreased
-
*
+
**no GDH2 RNA (y-axis) could be detected at ammonia concentrations of 29 and 44 mM
-
*
+
**this figure showed the concentration of ammonia both repressed and induced the RNA expression of nitrogen-regulated genes.
-
*
+
*GAP1 expression has been shown to be dependent on the ammonia concentration or flux
-
*
+
*Put4p has also been shown to be regulated in the same way
 +
*also in figure 2, at 29 and 44 mM ammonia the levels of GAP1 and PUT4 RNA remained constant
 +
**after, 44 mM the amounts of GAP1 and PUT4 RNA decreased and there was practically no GAP1 RNA at 118 mM ammonia, but there was still a small amount of PUT4 RNA
 +
**the figure showed that GAP1 and PUT4 is regulated by the ammonia concentration
 +
*Figure 2 also shows that the RNA in ILV5 and HIS4 increased and maximized at 66 mM of ammonia, but as the concentration continued to increase, the RNA expression decreased
====Enzyme Activities====
====Enzyme Activities====

Revision as of 02:11, 31 January 2013

Contents

Vocabulary

  1. biomass - Total mass of all living material in a specific area
  2. biosynthesis - The production of a complex chemical compound from simpler precursors in a living organism, usually involving enzymes (to catalyze the reaction) and an energy source
  3. flux - The total amount of a quantity passing through a given surface per unit time
  4. induced - To lead in; to introduce
  5. permase - The 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
  6. gram-negative bacteria - A bacteria which loses crystal violet stain but are stained pink when treated by Grams method
  7. metabolite - Any substance produced by metabolism or by a metabolic process
  8. glutamine - A crystalline amino acid occurring in proteins; important in protein metabolism.One of the 20 amino acidsthat are commonly found in proteins
  9. glutamate - A major fast excitatory neurotransmitter in the mammalian central nervous system
  10. transferase - A suffix to the name of an enzyme indicating that it transfers a specific grouping from onemolecule to another, for example acyl transferases transfer acyl groups

The definitions for these words were found at [Biology Online]

Outline

Introduction and Background Information

  • ammonia is the preferred source of nitrogen for Saccharomyces cerevisiae
  • nitrogen metabolism components are regulated at the level of enzyme activity and the level of gene expression
  • it is thought that the governing parameter of nitrogen metabolism is the ammonia flux, not the ammonia concentration
  • this experiment tested effects of different amounts of ammonia on gene expression and enzyme activities

Physiological Parameters

  • S. cerevisiae SU32 was grown with feeds containing different concentrations of ammonia
    • 29, 44, 61, 66, 78, 90, 96, 114, and 118 mM
  • they were also grown with a fixed glucose concentration of 100 mM at a dilution rate of 0.15 per hour
  • Figure 1A showed an increase of the ammonia concentration from 29 to 61 mM caused an increase of the biomass from 4.9 to 8.2 g/liter
    • with ammonia concentrations (x-axis) higher than 61 mM, the residual concentration of ammonia in the culture medium (y-axis) also increased to 62 mM but the biomass (y-axis) remained constant due to the fact that the glucose was now limiting
  • Figure 1B showed that an ammonia concentration (x-axis) greater than 44 mM resulted in the CO2 production (y-axis) and O2 consumption (y-axis) to remain constant.
    • input concentrations less than or equal to 44 mM, the values for CO2 produced and O2 consumed differed
      • ex: at 29 mM of ammonia, the CO2 production increased to 7.2 mmol/gh while the O2 consumption decreased to 1.5 mmol/gh
    • this figure shows that there is no real change in the carbon metabolism when the ammonia concentration is increased over 44 mM
  • Ammonia within the cell reacts with ketoglutarate to produce glutamate which is then converted to glutamine with another ammonium ion
  • Figure 1C showed the ketoglutarate concentration (y-axis) decreased from 10 to about 5 when the culture ammonia concentrations (x-acis) changed from limitation to excess
    • the intracellular glutamate concentration (y-axis) increased from about 75 to 220 and the glutamine concentration (y-axis) increased from approximately 4 to about 27

Northern Analyses

  • Northern Analyses were preformed see whether the RNA levels of nitrogen regulated genes changed with increasing ammonia concentrations
  • after being analyzed, the RNA data was quantified with X-ray films at different exposure times
  • Figure 2 showed that when the ammonia concentrations (x-axis) were increased up to 78 mM, the level of GDH1 RNA (y-axis) remained constant, but when it kept being increased the GDH1 RNA level decreased
    • no GDH2 RNA (y-axis) could be detected at ammonia concentrations of 29 and 44 mM
    • this figure showed the concentration of ammonia both repressed and induced the RNA expression of nitrogen-regulated genes.
  • GAP1 expression has been shown to be dependent on the ammonia concentration or flux
  • Put4p has also been shown to be regulated in the same way
  • also in figure 2, at 29 and 44 mM ammonia the levels of GAP1 and PUT4 RNA remained constant
    • after, 44 mM the amounts of GAP1 and PUT4 RNA decreased and there was practically no GAP1 RNA at 118 mM ammonia, but there was still a small amount of PUT4 RNA
    • the figure showed that GAP1 and PUT4 is regulated by the ammonia concentration
  • Figure 2 also shows that the RNA in ILV5 and HIS4 increased and maximized at 66 mM of ammonia, but as the concentration continued to increase, the RNA expression decreased

Enzyme Activities

Personal tools