Carmen E. Castaneda: Week 2

Preparation for Journal Club 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.

1. gene expression: The conversion of the information from the gene into mRNA via transcription and then to protein via translation resulting in the phenotypic manifestation of the gene. [1]
2. continuous cultures: A culture of microorganisms in a liquid medium which is maintained under constant conditions with a constant nutrient supply so that it can grow steadily for an extended period of time. [2]
3. ammonia assimilation: The utilization of ammonia (or ammonium ions) in the net synthesis of nitrogen-containing molecules; e.g., glutamine synthetase. [3]
4. steady-state concentration: When a concentration is at equilibrium[4]
5. ammonia flux: The total amount of ammonia passing through a given surface per unit time.[5]
6. biomass concentrations: Concentration of biological material used as a fuel, or source of [energy]. [6]
7. biosynthetic genes: Genes produced by biosynthesis[7] which is an organic reaction carried out that creates more complex molecules from simpler molecules.[8]
8. oligonucleotides: Polymers made up of a few (2-20) nucleotides, in molecular genetics, they refer to a short sequence synthesised to match a region where a mutation is known to occur, and then used as a probe.[9]
9. altered transcription: To alter, or be altered, in any manner; to suffer a partial changein transcription. [10]
10. posttranscriptional: Referring to events that occur after transcription[11], which is the process of transcribing or making a copy of genetic information stored in a DNA strand into a complementary strand of RNA (messenger RNA or mRNA) with the aid of RNA polymerases. [12]

Outline

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?

Abstract

Saccharomyces cerevisiae

• grown in a continuous culture
  • with input ammonia concentrations
    • nitrogen limitation
    • nitrogen excess
    • glucose limitation

Ammonia concentration might be the key factor in nitrogen metabolism instead of flux

Introduction

Saccharomyces cerevisiae rather ammonia for their nitrogen

• produces quicker growth
• able to regulate gene expression and level of enzyme activity
• through many experiments it has shown that the ammonia concentration plays a key role
  • differences exist between extrenal ammonia concentration and the rate of ammonia aasimililation
• Through this experiment they were looking at cultures with the same level of flux but different ammonia concentrations

Materials and Methods

• Saccharomyces cerevisiae SU32
• Ammonia Concentration 29,44,61,66,78,90,96,114,118 mM
• fixed glucose concentration of 100 mM
• amino acid permease encoding genes, GAP1 PUT4
• biosynthetic genes ILV5, HIS4, GDH1, GLN1, GS, ACT1,H2A-H2B

Physiological parameters

• They measured the ammonia and biomass concentrations

Northern Analyses

• checked to see if RNA levels of nitrogen regulated genes had changed according to the concentration of ammonia
• quantified data with X-ray films at different exposure times

Enzyme Activities

• investigated the changes in levels of activities as caused by the changes in ammonia concentrations
• measured the activities of NADPH-GDH and NAD-GDH under Vmax conditions

Results

Physiological parameters

• Ammonia Limitation was shown when an increase of the ammonia concentration in the feed from 29 to 61 mM

resulted in an increase of the biomass from 4.9 to 8.2 g liter21

• residual ammonia concentration shown to stay constant
  • feeds higher than 61mM showed an increase
• CO2 and 02 were produced at a constant rate
• culture conditions were changed when the ammonia concentration wen to excess form limitation

Northern Analyses

• The higher the concentration of ammonia, the level of GDH1 RNA stayed the same
  • but as more ammonia was added the level of GDH1 RNA began to decrease
• When the ammonia concentration was below 61mM there was no GDH2 RNA
  • but as it reached 61mM there was an increase of GDH2 RNA
• 61mM seemed to be when the most GNL1 occured and thus RNA of nitrogen-regulated genes was repressed and induced

Enzyme Activities

• when the ammonia concentration had increased, the activity of NADPH-GDH decreased
  • GDH1 decreased as well
• NAD-GDH incresead when ammonia was between 29-61 mM
  • as ammonia increased more, NAD-GDH did not increase
• GS began to decrease as the ammonia concentration increased
  • but after 61mM there was no change

It was concluded that the concentration of ammonia regulates the nitrogen metabolism of S. cerevisiae at different levels.

Discussion

Figure 1

A. Here we see the relationship between the NH4+ concentration vs the residual NH4+ concentration, which has a positivelinear relationship which shows that as NH4+ concentration so does the residual concentration. We also see NH4+ concentration vs biomass, a slight increase and NH4+ concentration vs NH4+ flux, constant relationship.

B.Here we see the relationship between NH4+ concentration vs the CO2 production, O2 consumption and Respiratory Quotient levels.

C.Here is the NH4+ concentration vs α-ketoglutarate, glutamate, and glutamine

Figure 2

Here we see the NH4+ concentration vs the % Expression levels of GDH1,GDH2 in the first graph, followed by the NH4+ concentration vs the %expression levels of GAP1 and PUT4, and finally NH4+ concentration vs % expression of GNL1, HIS4, ILV5.

Figure 3

Here we can witness the NH4+ concentration vs GS transferase as well as NH4+ concentration vs GS synthetase. Above this graph we se NH4+ concentration vs NAD-GDH and above that we see NH4+ concentration vs NADPH-GDH.

References

• Boles, E., W. Lehnert, and K. Zimmermann. 1993. The role of the NADdependent

glutamate dehydrogenase in restoring growth of a Saccharomyces cerevisiae phosphoglucose isomerase mutant. Eur. J. Biochem. 217:469–477.

• Courchesne, W. E., and B. Magasanik. 1983. Ammonia regulation of amino

acid permeases in Saccharomyces cerevisiae. Mol. Cell. Biol. 3:672–683.

• Dever, T. E., L. Feng, R. C. Weh, A. M. Cigan, T. F. Donahue, and A. G.

Hinnebusch. 1992. Phosphorylation of initiation factor 2a by protein GCN2 mediates gene specific translational control of GCN4 in yeast. Cell 68:585– 596.

• Magasanik, B. 1988. Reversible phosphorylation of an enhancer binding

protein regulates the transcription of bacterial nitrogen utilization genes. Trends Biochem. Sci. 13:475–479.

• Magasanik, B. 1992. Regulation of nitrogen utilization, p. 283. In J. R.

Broach, E. W. Jones, and J. R. Pringle (ed.), The molecular and cellular biology of the yeast Saccharomyces: gene expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

• Miller, S. M., and B. Magasanik. 1991. Role of the complex upstream region

of the GDH2 gene in nitrogen regulation of the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae. Mol. Cell. Biol. 11:6229–6247.

• Mitchell, A. P., and B. Magasanik. 1983. Purification and properties of

glutamine synthetase from Saccharomyces cerevisiae. J. Biol. Chem. 258:119– 124.

• Mitchell, A. P., and B. Magasanik. 1984. Three regulatory systems control

production of glutamine synthetase. Mol. Cell. Biol. 4:2767–2773.

• Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a

laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

• Sierkstra, L. N., E. G. ter Schure, J. M. A. Verbakel, and C. T. Verrips. 1994.

A nitrogen-limited, glucose repressed, continuous culture of Saccharomyces cerevisiae. Microbiology 140:593–599.

• Sierkstra, L. N., J. M. A. Verbakel, and C. T. Verrips. 1992. Analysis of

transcription and translation of glycolytic enzymes in glucose-limited continuous cultures of Saccharomyces cerevisiae. J. Gen. Microbiol. 138:2559–2566.

• 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 141:1101–1108.

• Wiame, J.-M., M. Grenson, and H. N. Arst, Jr. 1985. Nitrogen catabolite

repression in yeast and filamentous fungi. Adv. Microb. Physiol. 26:1–88