James P. McDonald Week 3

Biological Terms

1. Permease: "General term for a membrane protein that increases the permeability of the plasma membrane to a particular molecule, by a process not requiring metabolic energy." [[1]]
2. Isomerase: "An enzyme that converts molecules into their positional isomers." [[2]]
3. 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 (oligonucleotide probes)." [[3]]
4. Dehydrogenase: "Enzyme that oxidizes a substrate by transferring hydrogen to an acceptor that is either NAD/NADP or a flavin enzyme. An enzyme that is used to remove hydrogen from its substrate, which is used in the cytochrome (hydrogen carrier) system in respiration to produce a net gain of ATP." [[4]]
5. Synthetase: "Enzymes of class 6 in the e classification, catalyse synthesis of molecules, their activity being coupled to the breakdown of a nucleotide triphosphate." [[5]]
6. Biosynthetic: "Relating to or produced by biosynthesis." [[6]]
7. Glutamate: "Major fast excitatory neurotransmitter in the mammalian central nervous system." [[7]]
8. Glutamine: "A crystalline amino acid occurring in proteins; important in protein metabolism. One of the 20 amino acids that are commonly found in proteins." [[8]]
9. GAP1: "General amino acid permease, a gene found in Saccharomyces cerevisiae." [[9]]
10. PUT4: "Proline permease, a gene found in Saccharomyces cerevisiae." [[10]]

Outline

Introduction

• Saccharomyces cerevisiae was grown in various ammonia concentrations and the effects on the growth was observed.
  • A single dilution rate was using with a range of different ammonia concentrations.
  • The ammonia concentrations were varied to observe its effects on gene expression and enzyme activities.
• The main result of the study was that nitrogen metabolism is dependent on ammonia concentration, not its flux.

Significance

• Ammonia is the prefferred growth source of Saccharomyces cerevisiae as it results in faster growth.
  • Nitrogen metabolism is regulated by gene expression and enzyme activity.
• Previous research seems to show that ammonia concentration itself is the most important factor in nitrogen metabolism.
  • But, in these previous studies the cultures have differed in ammonium flux, leaving flux as the possible key factor.
  • This experiment uses cultures with the same level flux, only the ammonium concentrations fed in are different.

Methods

Physiological Parameters

• Saccharomyces cerevisiae SU32 was grown in continuous cultures
  • They were fed with different ammonia concentrations: 29, 44, 61, 66, 78, 90, 96, 114, 118 mM.
  • Contained a fixed glucose concentration at 100 mM.
  • Had a dilution rate of 0.15h^-1.
• Biomass and residual ammonia concentration were measured at the different ammonia concentrations.
• The ammonium flux was calculated using the biomass, ammonia concentration, and the residual ammonia concentration.
• The respiratory quotient was calculated using the measured values of CO₂ production and O₂ consumption at the different ammonia concentrations.
• Alpha-ketoglutarate, glutamate, and glutamine concentrations were measured at the different ammonia concentrations.

Northern Analyses

• RNA levels of nitrogen-regulated genes were observed to see if they changed in different ammonia concentrations.
  • Used amino acid permease-encoding genes: GAP1, PUT4 and biosynthetic genes: ILV5, HIS4.
  • P-labelled oligonucleotides were used to detect GDH1, GLN1, GAP1, ILV5, HIS4, ACT1, and H2A-H2B RNA levels and a separate oligonucleotide was used to analyze PUT4 RNA levels.
  • A P-labelled DNA fragment was used to detect GDH2 levels and H2A-H2B was used to detect GLN1 RNA levels.
  • Gene expression levels were detected using x-ray films and plotted to compare expression levels at different ammonia concentrations.

Enzyme Activities

• Investigated changes in enzyme activity in different ammonia concentrations.
  • Looked at enzymes involved in the conversion of ammonia into glutamate or glutamine: NADPH-GDH, NAD-GDH, and GS.

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