Individual Journal Week 11

Terms

• Batch-culture: A large-scale closed system culture in which cells are grown in a fixed volume of nutrient culture medium under specific environmental conditions (e.g. nutrient type, temperature, pressure, aeration, etc.) up to a certain density in a tank or airlift fermentor, harvested and processed as a batch, especially before all nutrients are used up. [1]
• Catabolite: product of catabolism, the breakdown of complex molecules into simpler ones. [2]
• Gene Regulatory Protein: Any protein that interacts with dna sequences of a gene and controls its transcription. [3]
• Up-regulation:process that increases ligand/receptor interactions due to an increase in the number of available receptors. [4]
• Down-regluation: the process that decreases ligand and receptor interactions or reduces the responsiveness of a cell to a stimulus following first exposure.This is often accompanied by an initial decrease in affinity of receptors for the agent and a subsequent reduction in the number of available receptors expressed on the surface which can result from internalisation of the ligand:receptor complex or from decreased expression of the receptor. [5]
• Motif: The smallest group of atoms in a polymer that, when under the influence of a rotation-translation operator, will assemble the rest of the atoms in the chain. [6]
• Desaturase: Any of several enzymes that putdouble bonds into the hydrocarbon areasof fatty acids. [7]
• Biogenesis: The process in which life forms arise from similar life forms. [8]
• Trehalose: a crystalline disaccharide C12H22O11 that is found in various organisms (as fungi and insects), is about half as sweet as sucrose, and is sometimes used as a sweetener in commercially prepared foods [9]
• Transcriptome: the complement of mature messenger RNAs produced in a given cell in a given moment of its life. [10]

Outline

Introduction

• The study observes the effects of suboptimal temperatures on transcriptional regulation in yeast.
• Transcriptional responses were observed at temperatures 12C and 30C.
• Lower temperatures, typically result in the slower cellular processes: growth phase, respiration, lipid composition of membranes, and trehalose content.
• Time scale of exposure to cold-shock relevant:
  • Sudden exposure => adaptation
  • Prolonged exposure=> acclimation
  • Long term exposure => evolutionary adaptation
• Two distinct phases during cold-shock response

1. First 12 hours, early cold shock (ECS)
2. After first 12 hours, Late cold response (LCR)

• TPS1 and TPS2, commonly observed trehalose-biosynthetic transcriptional induction genes- observed in cold and heat shock conditions
• Genes involved in cold-shock response typically involved in other stress response
• Growth rate an important factor on transcription
• Research seeks to control specific growth rate and other culture conditions, through use of chemostat, in order to establish steady-state and, thus, investigate transcription regulation influenced by suboptimal temperatures

Materials & Methods

• Yeast was grown in one chemostat with temperature 12C as well as a second chemostat set at 30C, both set at a dilution rate of 0.03h-1 with a working volume of 1.0 L
• Carbon or nitrogen limitations, other media/ nutrients in excess
• pH constant at 5.0 by addition of 2 M KOH
• Anaerobic growth
• Stirrer set at constant 600 rpm
• Grown in conditions in which ammonia was limiting at 12C, ammonia was limiting at 30C, glucose was limiting at 12C, and glucose was limiting at 30C
• Cultures analyzed through microarray and statistical analysis
• Microarray analysis:
  • Determined RNA quality
  • growth condition derived from three independently cultured replicates
  • Northern analysis used
• Statistical Analysis
  • Excel used to run significance of microarray add-in, through "pair-wise comparisons"
  • Data displayed in Venn diagrams and heat-maps
  • Promoters analyzed through use of web-based software
  • p-value calculated
  • Outside transcriptome datasets used for comparison

Results

• Table 1
  • At both 12C and 30C biomass yields and fermentation rates similar in both carbon and nitrogen limited chemostats
  • Therefore, growth efficiency not significantly affected by growth temperature
• Figure 1
  • Ven diagram displaying: Glucose limited cultures, 494 genes yielded significantly different transcript levels at both 12C and 30C
  • 806 genes yield significant transcription in nitrogen-limited culture
  • 16% of S. cervisiae genome temperature-responsive genes
  • Under both nutrient limitations, 235 genes demonstrated up or down regulation
• Figure 2
  • Heat map of 1065 different genes in anaerobic glucose and ammonia limited chemostat cultures at 12C and 30C
  • Change in transcript levels of genes involved in uptake of growth-limiting nutrients was reflected by changes in transport kinetics
  • Higher limiting nutrients at 12C resulted in higher catabolite repression
• Table 2
  • Displays protein and storage carbohydrates grown in ammonium and glucose limited anaerobic chemostat
  • In ammonium-limited chemostat, trehalose and glycogen significantly lower at lower temperature
• Table 3(A)
  • Msn2/Msn4 via STREs in their promoters regulate key genes relevant to storage carbohydrate synthesis
  • Overrpresentation of STRE elements in upstream regions of genes that showed a reduced transcript level at 12C in nitrogen limited chemostats
  • Consistent with stress induced glycogen and tehalose genes at low temps
• Table 3 (B)
  • Enrichment of PAC cis-regulatory motifs 5' upstream of sequences, increased transcript level at 12C
  • Transcription Factors: Mbp1p, Hap2-Hap1, Hap3-Hap1, Fhl1p, Sfp1p, Gln3p, Gln3-Dal82, Hap2-dal82, Aft2p, Hsflp, Nrg1p, Phd1p, Rsc1p, Sok2p, Nrg1-Aft2, Phd1-Nrg1, Rox1-Phd1, Sok2-Nrg1
• Figure 3
  • Displays genes that are common to the three batch-culture studies on transcriptional adaptations in relation to low temperatures
  • Heat map displays transcript ratio of 259 genes found in common in the three batch-culture low temp transcriptome data sets.
• Figure 4
  • 235 genes that displayed transcription changes resulting from acclimation were compared to 259 low-temperature adaptation genes.
  • In total there were 11 genes that displayed a pattern of regulation in all datasets.
• Figure 5
  • Through the use of Venn diagrams looking at adaptation and acclimation, up or down regulated genes are compared.
  • Overlap was considered "negligible" between growth-rate-responsive genes in comparison to temperature-responsive genes.
• Figure 6
  • Compared to environmental stress, 233 genes show an opposite transcriptional response to low temperature
  • ESR is, thus, a response that occurs during sudden exposure to suboptimal temperatures