Anthony J. Wavrin Week 3

Introduction

• This article is exploring one of the possible explanations of how nitrogen, used in the form of ammonia in this study, can effect Saccharomyces cerevisiae.
• It is well known that nitrogen is an essential nutrient that can increase growth, as utilized in fertilizer.
• It is hypothesized that the actual influx of nitrogen may cause growth instead of the concentration.
• This study tests explores if increasing concentration of ammonia while keeping a constant influx will cause nitrogen related responses.
• The concentrations used resulted in testing from nitrogen limitation to nitrogen excess.
• Overall, they conduct effects of physiological parameters, RNA expression, and enzyme activites.

Physiological parameters

• The concentrations of ammonia used were 29, 44, 61, 66, 78, 90, 96, 114, and 118mM.
  • It is interesting to note that at the concentration of 61mM of ammonia, glucose becomes the limiting nutrient.

Figure 1

A

• The X-axis represents the increase in the concentration of the ammonia.
• The Y-axis on the left represents the residual ammonia concentration.
• The Y-axis on the right represents the biomass (dry weight).
• The Y-axis on the far right represents the ammonia flux, which is calculated using ammonia concentration, residual ammonia concentration, and biomass.
• As ammonia increased to ammonia saturation, there was an increase in biomass, but stayed relatively constant after ammonia excess (>61mM).
• The residual ammonia concentration sky rockets after 61mM which is expected due to nitrogen excess.

B

• The X-axis represents the increase in the concentration of the ammonia.
• The Y-axis on the left represents the CO2 production.
• The Y-axis on the far left represents the O2 usage.
• The Y-axis on the right represents the respiratory quotient, which is CO2 production/ O2 usage.
• Concentrations above 44mM of ammonia have a relatively flat respiratory quotient, indicating carbon is the limiting nutrient.

C

Left Panel

• The X-axis represents the increase in the concentration of the ammonia.
• The Y-axis on the left represents the concentration of α-ketogluterate present.
• As the ammonia concentration increases, α-ketogluterate concentration decreases until 61mM. This represents the conversion of α-ketogluterate with nitrogen to form glutamate and eventually glutamine.

Middle Panel

• The X-axis represents the increase in the concentration of the ammonia.
• The Y-axis on the left represents the concentration of glutamate present.
• As the ammonia concentration increases, glutamate concentration increases until 61mM.

Right Panel

• The X-axis represents the increase in the concentration of the ammonia.
• The Y-axis on the left represents the concentration of glutamine present.
• As the ammonia concentration increases, glutamine concentration increases continually.

RNA Expression

• To measure the levels of RNA of nitrogen related genes, northern analysis was performed.
• RNA was detected using labeled phosphate oligonucleotides or 2.5kB Xho1-Bam1 DNA fragments.
• X-ray films were utilized to quantify the RNA Detected.

Figure 2

Left Panel

• The X-axis represents the increase in the concentration of ammonia.
• The Y-axis represents the % expression of a given RNA, using ACT1 and H2A-H2B as internal controls.
• As the ammonia concentration increases past 61mM, GDH1 decreases while GDH2 increases, indicating that GDH1 is repressed by excess nitrogen while GDH2 is induced by excess nitrogen.

Middle Panel

• The X-axis represents the increase in the concentration of ammonia.
• The Y-axis represents the % expression of a given RNA, using ACT1 and H2A-H2B as internal controls.
• Both GAP1 and PUT4 "peak" and 44mM and decrease til 78mM then become relatively stable, indicating that they are repressed by excess nitrogen.

Right Panel

• The X-axis represents the increase in the concentration of ammonia.
• The Y-axis represents the % expression of a given RNA, using ACT1 and H2A-H2B as internal controls.
• GLN1, HIS4, and ILV5 all peak around 66mM and 78mM and stay at a high expression until the highest concentration (118mM), in which the expression decreases drastically.