James C. Clements: Week 2

James C. Clements

BIOL398-01/S11:Assignments

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Assignment Prompt - Preparation for Journal Club 1

1. 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.
2. 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?

Response

Vocab

• Acetaldehyde - a colourless, flammable liquid used in the manufacture of acetic acid, perfumes, and flavors. It is also an intermediate in the metabolism of alcohol. [1]
• Acetate - a salt or ester of acetic acid [2]
• Amino acid starvation - lengthy and continuous deprivation of amino acids [3]
• Biosynthetic gene - A gene related to the building up of a chemical Compound in the physiologic processes of a living organism [4]
• Gram-negative bacteria - bacteria which lose crystal violet stain but are stained pink when treated by grams method [5]
• Northern Analysis (blot) - An electroblotting method in which rNA is transferred to a filter and detected by hybridisation to (32)P labelled RNA or dNA. [6]
• Oligonucleotide - linear sequence of up to 20 nucleotides joined by phosphodiester bonds [7]
• Proline - One of the 20 amino acids directly coded for in proteins [8]
• Reductoisomerase - an enzyme that catalyzes a reduction reaction and catalyzes its substrate to an isomeric form [9] [10]
• Standard Error of Mean - The standard deviation of a data set or of the population divided by the square root of the sample size [11]

Outline of Article

Abstract

1. Experiment - Saccharomyces Cerevisiae (baker's yeast) was grown at a single dilution rate with varying concentrations of input ammonia
2. What Happened?
   • Ammonia assimilation stayed approximately constant
   • Increased ammonia resulted in:
     • Increased: glutamate, glutamine, levels of NAD activity and mRNA
     • Decreased: NADPH activity and mRNA, mRNA for production of GAP1 and PUT4
3. Conclusion - Governing factor of nitrogen metabolision might be concentration of ammonia instead of flux

Introduction

1. What is known
   • Ammonia is a preferred nitrogen source for Saccharomyces cerevisiae
     • Nitrogen metabolism regulated both by gene expression and level of enzyme activity
     • Ammonia concentration is a key influence
2. What is unknown
   • Is concentration really a key influence or is the flux of nitrogen a key influence
3. Experimental approach
   • Keep flux of ammonia constant and vary concentration to see if concentration is a key influence

Physiological Parameters

1. Focus: Intracellular concentrations
2. Method: S. cerevisiae SU32 grown in continuous cultures with feeds containing
   • Varying ammonia concentrations
   • Fixed glucose concentration
3. Figure 1 A
   • Increase of ammonia concentration from 29 to 119 mM. Investigating Residual concentration, biomass, and flux
     • Data interpretation:
       • Increase of biomass from 4.9 to 8.2 g/L when ammonia concentration varies from 29 to 61 mM
       • Residual ammonia concentration in culture was constant at abot 0.022 mM when ammonia concentration varies from 29 to 61 mM
       • Risidual ammonia concentration in culture increased linearly when concentration larger than 61 mM
   • Observation: Glucose became limiting factor at concentrations greater than 61 mM
4. Figure 1 B: Investigating Oxygen Consumption, Carbon Dioxide consumption, and the Respitory quotient
   • Data interpretation:
     • Respiratory quation nearly constant when more than 44 mM ammonia present
     • Ammonia limitation of con. less than 44 mM - quotient varies decreases as con. increases
5. Figure 1 C: Concentration of alpha-Ketoglutarate, Glutamate, and Glutamine vs. ammmonia concentration
   • Data Interpretation as ammonia concentration increases:
     • alpha-Ketoglutarate decreases
     • Glutamate increases
     • Glutamine increases

Northern Blot

1. Focus: RNA levels
2. Method: Northern blots performed to investigate whether the RNA levels of regulated genes changed with ammonia concentrations
   • Amino acid permease-encoding genes: GAP1, PUT4
   • Biosynthetic genes: ILV5 (a-acetoacetate reductoisomerase), HIS4 (histidinol dehydrogenase)
   • All except PUT4 RNA levels detected with 32P-labelled oligonucleotides
   • PUT4 analyzed using oligonucleotide: 5'-CTCCTCCTTCTTGGTGTCGCCGCCGCTACC-3'
3. Figure 2: RNA expression of nitrogen-regulated genes at different ammonia concentrations in the feed
   • Interpretation of effect of increased ammonia concentration:
     • GDH1 decrease
     • GDH2 increases overall but oscillates at higher concentrations
     • GAP1 decreases after an initial increase
     • PUT4 decreases after an initial increase
     • GLN1 increases and then decreases
     • HIS4 increases and then decreases
     • ILV5 increases and then decreases

Enzyme Activities

1. Focus: affect of increased ammonia concentrations on levels of enzyme activity
2. Method: Levels of NADPH-GDH, NAD-GDH, GS activity determined at varying concentrations
3. Figure 3: In vitro activity levels of NADPH-GDH, NAD-GDH, GS transferase, GS synthetase
   • Observations as ammonia concentration increases:
     • NADPH-GDH decreases
     • NAD- GDH increases
     • GS transferase decreases
     • GS synthetase decreases

Conclusion

1. Concentration of ammonia regulates nitrogen metabolism of Saccharomyces cervisiae
2. Regulation is due to either extracellular or intracellular concentrations of ammonia or levels of intracellular metabolites
3. Saccharomyces cervisiae could have an ammonia sensor as has been found in some bacteria

Shared Journal Questions and Answers

1. What was the purpose of this assignment? - To become familiar with the article which will soon be discussed; to learn any unknown terms that it uses and to gain exposure to the methodology. (Perhaps so that we can eventually model this system?)
2. What aspect of this assignment came most easily to you? - finding definitions for the the vocabulary; not all of the terms were readily available, but it wasn't too difficult to understand the terms after some minor reading.
3. What aspect of this assignment was the most challenging for you? - outlining the article: I understand the format that was used was devised to split things up into categories, but it made certain aspects of the paper difficult to keep track of.
4. What (yet) do you not understand?
   • Why was this published in the Journal of Bacteriology? This doesn't seem like a very suitable place for an article about eukaryotic acquisition of nitrogen
   • Also the data points for glutamate and glutamine have a sharp drop on the last data point. What could have caused this?