Paul Magnano:Week 3 Individual Journal: Difference between revisions
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*RNA analyses were done to see if RNA levels of nitrogen regulating genes changed with increased ammonia concentrations | *RNA analyses were done to see if RNA levels of nitrogen regulating genes changed with increased ammonia concentrations | ||
*genes GDH1, GDH2,GLN1 were determined to study responses to ammonia levels | *genes GDH1, GDH2,GLN1 were determined to study responses to ammonia levels | ||
*GAP1 and PUT4 are amino acid permease genes | |||
*biosynthetic genes were ILV5 and HIS4 | |||
*Fig 2 | *Fig 2 | ||
**increasing ammonia levels resulted in GDH1 levels staying the same | **increasing ammonia levels resulted in GDH1 levels staying the same | ||
**ammonia concentrations of 29-44mM there was no GDH2, but with increase to 61mM GDH2 level increased | **ammonia concentrations of 29-44mM there was no GDH2, but with increase to 61mM GDH2 level increased | ||
**maximum GLN1 expression was observed at 61mM | **maximum GLN1 expression was observed at 61mM | ||
** overall it was seen that ammonia concentration both repressed GDH1 and caused the expression of GDH2 | **overall it was seen that ammonia concentration both repressed GDH1 and caused the expression of GDH2 | ||
**GAP1 was shown to be regulated in response to ammonia concentration | |||
**the influence of ammonia concentration on GAP1 and PUT4 was analzyed in this study | |||
**when ammonia concentration was above 44mM, GAP1 and PUT4 decreased | |||
**in ammonia limited culture a relationship between ammonia flux GAP1 expression wa observed | |||
**GAP1 and PUT4 expression are regulated by ammonia flux not by ammonia concentration | |||
**genes ILV5 and HIS4 increased with increasing ammonia concentration outside the cell, it was highest at 66mM | |||
===Enzyme Activities=== | |||
==Ten New Terms== | ==Ten New Terms== |
Revision as of 20:19, 30 January 2013
Outline
Experiment
- sacchromyces cerevisiae was grown in continuous culture, with varying ammonia input concentrations, while ammonia assiilation rates remained constant
- ammonia is a preffered nitrogen source for s. cerevisiae, because it results in faster growth than other sources
- in this study, cultures were grown with same flux (relationship b/w external ammonia concentration and rte of assimilation) but different inflow ammonia concentrations
- Physiological parameters:
- S. cerevisiae SU32 grown in continuous cultures with ammonia inflow concentrations ranging from 29 to 118mM
- fixed glucose concentration of 100mM
- Fig 1A
- increase of ammonia concentration from 29 to 61mM resulted in increase of biomass from 4.9 to 8.2 g/liter
- ammonia concentration above 61mM biomass remained at 8.2 g/liter
- over trhe entire range of ammonia concentrations the ammonia flux into biomass was 1.1 mmol/gh
- Fig 1B
- input ammonia concentration above 44mM resulted in (c02 production/02 consumption) remained constant
- with ammonia limitation (ammonia input below 44mM) c02 production and 02 consumption values differed
- no changes in the residual glucose concentration were seen
- no significant changes in carbon metabolism occured when the culture was switched from ammonia excess to ammonia limitation
- Fig 1C
- ammonia within the cells reacts with ketoglutarate to produce glutamate which is converted to glutamine
- ketoglutarate concentration decreased from 10 to 5 umol/g when the ammonia concentration changed from limited to excess
- glutamate concentration increased within the cell, linearly
- increaing ammonia concentrations caused the concentrations of glutamate and glutamine within the cell to increase
Northern Analyses
- RNA analyses were done to see if RNA levels of nitrogen regulating genes changed with increased ammonia concentrations
- genes GDH1, GDH2,GLN1 were determined to study responses to ammonia levels
- GAP1 and PUT4 are amino acid permease genes
- biosynthetic genes were ILV5 and HIS4
- Fig 2
- increasing ammonia levels resulted in GDH1 levels staying the same
- ammonia concentrations of 29-44mM there was no GDH2, but with increase to 61mM GDH2 level increased
- maximum GLN1 expression was observed at 61mM
- overall it was seen that ammonia concentration both repressed GDH1 and caused the expression of GDH2
- GAP1 was shown to be regulated in response to ammonia concentration
- the influence of ammonia concentration on GAP1 and PUT4 was analzyed in this study
- when ammonia concentration was above 44mM, GAP1 and PUT4 decreased
- in ammonia limited culture a relationship between ammonia flux GAP1 expression wa observed
- GAP1 and PUT4 expression are regulated by ammonia flux not by ammonia concentration
- genes ILV5 and HIS4 increased with increasing ammonia concentration outside the cell, it was highest at 66mM