Sarah Carratt: Week 2
- 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.
- 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.
- What do the X and Y axes represent?
- How were the measurements made?
- 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?
- Each group of students will be assigned one section of the paper. The group will be responsible for explaining the section, including any tables/figures in detail to the class. Groups will be assigned on 1/20/11 in class. Dr. Dahlquist will prepare the PowerPoint slides this time; for future journal club assignments, you will prepare the PowerPoint.
- Physiological parameters section, Figure 1: James, Nick
- Northern analysis section, Figure 2: Carmen, Alondra
- Enzyme activities section, Figure 3: Sarah
- Article: The Concentration of Ammonia Regulates Nitrogen Metabolism in Saccharomyces cerevisiae
- Biology Dictionary
- Gene Ontology
- NCI Dictionary of Cancer Terms
Sarah Carratt 21:09, 23 January 2011 (EST)
Terms and Definitions
- ammonia assimilation: The utilization of ammonia (or ammonium ions) in the net synthesis of nitrogen-containing molecules; e.g., glutamine synthetase. 
- residual: Remaining or left behind. 
- proline: One of the 20 amino acids directly coded for in proteins. 
- biosynthetic: Relating to or produced by biosynthesis, which is the creation of more complex molecules from simpler molecules, such as the conversion of glucose to starch. 
- gram-negative bacteria: Bacteria which lose crystal violet stain but are stained pink when treated by grams method. 
- gram-positive bacteria: Bacteria that retain the stain or that are resistant to decolourisation by alcohol during grams method of staining. 
- flux: The total amount of a quantity passing through a given surface per unit time. Typical quantities include (magnetic) field lines, particles, heat, energy, mass of fluid, etc. 
- biomass:The total mass of all living material in a specific area, habitat, or region. 
- parameter: A variable whose measure is indicative of a quantity or function that cannot itself be precisely determined by direct methods, for example, blood pressure and pulse rate are parameters of cardiovascular function and the level of glucose in blood and urine is a parameter of carbohydrate metabolism. 
- quantification:The expression of a numerical amount. 
- "Saccharomyces cerevisiae" was grown in cultures with various amounts of ammonia.
- While the uptake of ammonia proved to be constant, there was a difference in net output when the culture was given more ammonia.
- Increases in ammonia resulted in:
- Higher concentrations of intracellular glutamate and glutamine
- Increases in levels of NAD-dependent glutamate dehydrogenase activity and its mRNA (gene GDH2)
- Decreases in levels of NADPH-dependent glutamate dehydrogenase activity and its mRNA (gene GDH1)
- Decreases in the levels of mRNA for the amino acid permease-encoding genes GAP1 and PUT4
- Nitrogen metabolism is likely to be based on the abundance of ammonia and the ability to consume the ammonia longer because of its excess.
- Nitrogen metabolism is NOT likely to be based on a change in the rate of consumption because of excess ammonia.
- "Saccharomyces cerevisia" get nitrogen from ammonia.
- Ammonia allows for better growth when compared to proline or urea.
- Other Research:
- Ammonia concentration is the thing to focus on.
- Important variables to consider:
- External ammonia concentration
- Rate of ammonia assimilation (i.e., the ammonia ﬂux)
Materials and Methods
- Yeast was grown in cultures with:
- Ammonia concentrations of 29, 44, 61, 66, 78, 90, 96, 114, and 118 mM
- Glucose concentration of 100 mM
- Dilution rate of 0.15 h21
- Measurements were taken of ammonia concentrations and biomass.
- Calculated ammonia flux:
- dilution rate x (input ammonia concentration - residual ammonia concentration)/biomass
- Northern (RNA) analyses
- Did nitrogen-regulated genes change with increasing ammonia concentrations?
- Mitchell and Magasanik methods:
- NADPH-GDH and NAD-GDH measured under Vmax conditions
- GS activities were analyzed
- Northern (RNA) analyses
- The Numbers!
- Concentration Increase: 29 -> 61 mM
- Resulting Change in Biomass: 4.9 -> 8.2 g/L
- Residual Concentration: 0.022 mM (no change)
- In excess ammonia of 61 mM, biomass stayed at a constant 8.2 g/L.
- Residual concentration increased linearly.
- At concentrations under 44 mM, increased CO2 production of 7.22 mM/g and reduction in O2 consumption of 1.5 mM/g.
- Suggests alternative metabolic pathways being used because of presence of metabolites at low ammonia concentrations.
- Concentration of ammonia controls the nitrogen metabolism of “S. cerevisiae”
- Results are known because:
- Ammonia consumption rate remained constant
- Concentration of ammonia in the feed increased
- Possibilities for regulation:
- Extracellular or intracellular concentrations of ammonia
- Changes in levels of intracellular metabolites like a-ketoglutarate, glutamate, or glutamine.
- If the ammonia concentration is the regulator…
- “S. cerevisiae” might have an ammonia sensor which could be a two-component sensing system for nitrogen
- “S. cerevisiae” might be similar to gram-negative bacteria
- If the ammonia concentration is the regulator…
- In this experiment, the scientists grew budding yeast in ammonia and measured the net differences that resulted from varying this amount of ammonia. The results were that nitrogen metabolism is correlated with ammonia abundance and increases in nitrogen can likely to be attributed to the yeast's ability to infinitely consume ammonia at a constant rate. Nitrogen metabolism is NOT likely to be based on a change in the rate of consumption because of excess ammonia.
- Because yeast is a model organism, the significance of this work lies in the understanding of other living things and our ability to manipulate genes and environmental conditions to get the results we desire.
- In previous studies, the external ammonia and ammonia assimilation were combined into one variable and the possibility of a flux was not able to be tested.
- They used the northern (RNA) analyses and methods outlined by Mitchell and Magasanik.
- Results in the figures:
- FIGURE ONE
- A: This figure shows the relationships between ammonia and biomass concentrations. The x-axis is ammonia concentrations. The y-axis is residual ammonia concentration, ammonia flux, and biomass in mM/g. The residual ammonia concentration is constant after 60 mM, while the biomass keeps increasing when the ammonia concentration increases.
- B: This figure shows the relationships between ammonia concentration and O2/CO2. The x-axis is ammonia concentrations. The y-axes are O2 consumption, CO2 production, and the respiratory quotient. The general trend is that as ammonia concentration increases, O2 consumption increases and both the respiratory quotient and the CO2 production decrease. The respiratory quotient settles at about 42 mM, O2 at 20mM, and CO2 production settles at about 60 mM and then stays constant.
- C: This figure shows the relationship of ammonia concentrations and α-ketogluterate, glutamate and glutamine. The x-axis is ammonia concentrations. The y-axes is labeled for levels of α-ketogluterate, glutamate and glutamine. The general trend is that as ammonia concentrations increase, α-ketoglutarate decreases, but glutamate and glutamine increase.
- FIGURE TWO
- This figure shows ammonia concentrations as related to nitrogen regulated genes. The x-axis is ammonia concentrations. The y-axis is the percent expression of nitrogen regulated genes (RNA). When ammonia concentrations increase, then GDH1, GAP1 and PUT4 decrease and GDH2, HIS4, ILV5 and GLN1 increase.
- FIGURE THREE
- This figure shows ammonia concentrations as related to in vitro activity levels of NADPH-GDH, NAD-GDH, GS transferase. The x-axis is ammonia concentration. The y-axis is the GS transferase, NAD-GDH, and NADPH-GDH levels. As ammonia concentrations increase, NADPH-GDH decreases and NAD-GDH increases, while GS transferase is constant after 60 mM.
- FIGURE ONE
- Overall, nitrogen metabolism is not likely to be based on an increased assimilation of ammonia, but on the general concentration of ammonia that is present.