Lucia I. Ramirez Week 2
- ammonia: The common Name for NH3, a strongly basic, irritating, colourlessgas which is lighter than air and readily soluble in water. It is formed in nature as a by-product of protein metabolism in animals.Industrially, it is used in explosives, fertiliser, refrigerants, household cleaningsolutions, etc. (biology online.org. (2005). biology online: Answers to all your Biology Questions. Retrieved from http://www.biology-online.org/dictionary/Ammonia on 26 January 2015.)
- glutamate: major fast excitatory neurotransmitter in the mammalian central nervous system. (biology online.org. (2005). biology online: Answers to all your Biology Questions. Retrieved from http://www.biology-online.org/dictionary/Glutamate on 26 January 2015.)
- culture medium: a substance, either solid or liquid, used for the cultivation, isolation, identification, or storage of microorganisms.(biology online.org. (2005). biology online: Answers to all your Biology Questions. Retrieved from http://www.biology-online.org/dictionary/Culture_medium on 26 January 2015.)
- residual: Remaining or left behind. (biology online.org. (2005). biology online: Answers to all your Biology Questions. Retrieved from http://www.biology-online.org/dictionary/Residual on 26 January 2015.)
- 32P-labelled oligonucleotides: Replica filters were probed with 32 P-labeled cDNA prepared from polyadenylated RNA extracted from sexually induced or asexually growing V. carteri spheroids. Hybridization was performed according to standard procedures (Sambrook et al., 1989). Nucleotide sequences were determined by the dideoxy chain termination method (Sanger et al., 1977). Synthetic oligonucleotides were used to sequence clone A and clone B cDNAs in both directions. The sequence data have been deposited in the GenBank database under accession numbers AF058716 (clone A) and AF058717 (clone B). (biology online.org. (2005). biology online: Answers to all your Biology Questions. Retrieved from http://www.biology-online.org/articles/sex-inducing_pheromone_wounding_trigger/methods.html on 26 January 2015.)
- components of nitrogen metabolism are regulated at both the level of gene expression and the level of enzyme activity
- key influence of the ammonia concentration itself
- conceivable that the governing parameter is the ammonia flux itself rather than the concentration
- Main Point:
- discriminate between cultures having same levels of flux but having feeds with different ammonia concentrations
- Physiological parameters
- S. cerevisiate SU32 was grown in continuous cultures with feeds containing different ammonia concentrations and a fixed glucose concentration
- ammonia and biomass concentrations were “measured by using the L-glutamic acid determination kit” (ter Schure, E. G., H. H. W. Sillje, L. J. R. M. Raeven, J. Boonstra, A. J. Verkleij, and C T. Verrips. 1995, Nitrogen-regulated transcription and enzyme activities in continuous cultures of Saccharomyces cerevisiae. Microbiology 141:1101)
- Figure 1A
- Shows an increase of ammonia concentration in the feed resulted in an increase of the biomass showing ammonia limitations
- under conditions with a concentration of more than 61mM ammonia in the feed, glucose became limiting
- from the biomass, the ammonia concentration in the feed, and the residual ammonia concentration, the ammonia flux cab
- Figure 1B
- shows that at an input ammonia concentration above 44mM, the CO2 production and O2 consumption,
- indicate that, except at 29mM ammonia in the feed, no significant changes in the carbon metabolism occurred when the ammonia concentration in the feed was ***increased and the culture was switched from ammonia limitation to ammonia excess.
- Methods used to analyze the effects of the increasing ammonia concentrations on the intracellular alpha-ketoglutarate, glutamate, and glutamine concentrations.
- Sampling of the culture medium was performed by quick filtration of the culture liquid through a 0.22 um membrane filter.
- Figure 1C
- the alpha-ketoglutarate concentration decreased when the culture conditions changed from ammonia limitation to ammonia excess, and the intracellular glutamate concentration increased.
- with constant ammonia flux but increasing ammonia concentrations, the intracellular concentrations of glutamate and glutamine increase.
- Northern analyses.
- formed to investigate whether the RNA levels of nitrogen-regulated genes changed with increasing ammonia concentrations
- the expression levels of genes involved in the utilization of ammonia, GDH1, GDH2, and GLN1, were determined to study their responses to the amount of ammonia.
- Figure 2
- shows ammonia concentrations in the x-axis and the levels of GDH1 RNA and GDH2 RNA in the y-axis
- the concentrations of ammonia both repressed (GDH1) and induced (GDH2) the RNA expression of nitrogen-regulated genes.
- GAP1 and PUT4 expression are regulated not by the ammonia flux but by the ammonia concentration
- Enzyme activites
- Find if the changed ammonia concentrations resulted in changes in the levels of activity of enzymes involved in the conversion of ammonia into glutamate or glutamine
- Figure 3
- Shows ammonia concentration as x-axis and the levels of NADPH GDH, NAD-GDH, and GS as the y-axis.
- a further increase in the ammonia concentration did not result in a further increase in the level of NAD-GDH activity
- strongly suggested that NAD-GDH is regulated mainly at level of transcription
- altered level of GS activity was not caused by an altered transcription; therefore posttranscriptional.
- The data shows that the concentration of ammonia regulates the nitrogen metabolism of S. cerevisiae on various levels.
- Changes in nitrogen metabolism of S. cerevisiae are regulated by either extracellular or intracellular concentrations of ammonia or by changes in levels of intracellular metabolites
- Implications: S. cerevisiae has an ammonia sensor which could be a two-component sensing system for nitrogen.
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