Lauren M. Magee Week 2
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- permease: Any of several enzymes that transport membrane proteins.(Source:https://glosbe.com/en/en/permease)
- assimilation: The process of adapting or adjusting to the culture of a group or nation. (Source: http://dictionary.reference.com/browse/assimilation)
- extracellular: Occurring or being (situated) outside the cell or cells. (Source: http://www.biology-online.org/dictionary/Extracellular)
- intracellular:Occurring or being (situated) inside a cell or cells. (Source: http://www.biology-online.org/dictionary/Intracellular)
- flux: Occurring or being (situated) inside a cell or cells. (Source: http://www.biology-online.org/dictionary/Flux)
- biomass: The total mass of living matter within a given unit of environmental area. (Source: https://dictionary.search.yahoo.com/search;_ylt=A86.J7r6RMdUvVEAFYInnIlQ;_ylu=X3oDMTB0ZWVkYm84BHNlYwNzYwRjb2xvA2dxMQR2dGlkA1lIUzAwMl8x?p=biomass&.sep=)
- parameters: One of a set of measurable factors that define a system and determine its behavior and are varied in an experiment. (Source: https://dictionary.search.yahoo.com/search;_ylt=AwrTca9CRcdULZUAr70nnIlQ;_ylu=X3oDMTB0ZWVkYm84BHNlYwNzYwRjb2xvA2dxMQR2dGlkA1lIUzAwMl8x?p=parameter&.sep=)
- cultured: General term referring to the maintenance of cell strains or lines in the laboratory. (Source: http://www.biology-online.org/dictionary/Cell_culture)
- transferase: A suffix to the name of an enzyme indicating that it transfers a specific grouping from one molecule to another, for example acyl transferases transfer acyl groups. (Source: http://www.biology-online.org/dictionary/Transferase)
- gram-negative: Of, or relating to the group of bacteria that take the color of the counterstain (i.e. pink) under the microscope following gram staining. (Source: http://www.biology-online.org/dictionary/Gram-negative)
- Ammonia is a preferred nitrogen source for Saccharomyces cerevisiae. Nitrogen metabolism is regulated at both the level of gene expression and the level of enzyme activity. Research has pointed out the influence of the concentration of ammonia and the rate of ammonia assimilation. Because of this interaction, the researchers hypothesized that the ammonia flux may have a larger impact than the concentration of ammonia in nitrogen metabolism. To study this, the researchers observed cultures having the same levels of flux but having feeds with different ammonia concentrations.
Material & Methods
- S. cerevisiae was used to observe the effects of differing ammonia concentrations gene expression and enzyme activities. S. cerevisiae was continuously cultures and then feed ammonia in concentrations ranging from 29 to 118 mM along with a fixed glucose concentration and dilution rate.
Results & Discussion
- From Figure 1A, it can be determined that there was an increase in the biomass from 4.9 to 8.2 g/liter when the concentration of ammonia changed from 29 to 61 mM depicting ammonia limitation. In concentrations above 61mM, the residual ammonia concentration in the culture appears to be increasing linearly, while the concentrations below 61mM appeared to have a constant ammonia residual. The biomass for the concentrations above 61mM, however maintained 8.2 liters and the glucose became the limiting factor. Over the entire range of ammonia concentrations, the flux was about 1.1 mmol g^(-1) h^(-1).
- From Figure 1B, we can observe that in cultures with ammonia concentrations of 44mM or above, the respirations quotient remained constant. This constant was calculated by dividing the amount of carbon dioxide produced by the amount of oxygen consumed. However, when the ammonia concentration was at or lower than 44mM, the respirations ratios differed. There we no changes in the residual glucose concentration and as the cultures change from ammonia limitation to ammonia excess there appeared to be no effect on carbon metabolism.
- From Figure 1C, we are provided information on the effects of ammonia concentration on the intracellular concentrations of alpha-ketoglutarate, glutamate, and glutamine. Alpha-ketoglutarate decreased when the culture changes from one of ammonia limitation to ammonia excess. At a certain level of higher ammonia concentration, both glutamate and glutamine increased in their intracellular concentration.
- Northern RNA analysis of nitrogen-regulated genes was performed to observe the effects ammonia concentrations had on RNA levels. Two amino acid permease encoding genes were included, as well as nine biosynthetic genes. RNA levels were detacted with P-labelled oligonucleotides. The data was then quantified with X-ray films at different exposure time.
- Figure 2A shows the changes in ammonia concentration both repressed (GDH1) and induced (GDH2) the RNA expression of nitrogen-regulated genes. Figure 2B depicts the GAP1 and PUT4 genes, which appear not to be regulated by ammonia flux, but by changes in ammonia concentration. Finally, Figure 2Cshows biosynthetic genes ILV5 and HIS4. With the increasing of extracellular ammonia concentrations, both genes increased in their expression. However after reaching a concentration of 66mM the levels begin to decrease. The x-axis on these graphs is the ammonia concentration of the culture and the y-axis is the percentage of gene expression for each gene studied.
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