Ashley Rhoades Week 11

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Contents

Terms

  • In vivo: Taking place in a living organism
  • Cuvette:A straight-sided, optically clear container for holding liquid samples in a spectrophotometer or other instrument.
  • Transcriptome:The transcriptome is the set of all RNA molecules
  • Assay:Determine the content or quality of
  • Trehalose:a crystalline disaccharide C12H22O11
  • Ergosterol: A steroid alcohol in ergot and other fungi, converted to vitamin D2 when irradiated with ultraviolet light.
  • Supernatant: Denoting the liquid lying above a solid residue after crystallization, precipitation, centrifugation, or other process.
  • Mannoproteins: Component of yeast cell walls; protein covalently linked to polymers of mannose
  • catabolite: A product of catabolism.
  • orthologue:A gene in two or more species that has evolved from a common ancestor.

Outline

  • Introduction
    • temperatures below the optimal range for Saccharomyces cerevisiae slow down enzymatic reactions and affect many cellular processes
    • If exposure to a temperature change is sudden there will be a stress response
    • Experiments have shown two phases of a col shock response; an early cold response, and a late cold response
    • Experiments on the yeast cold shock response often have contradicting results.
    • Genes that show responses to cold shock includes TPS1, TPS2, HSP12, HSP26, HSP42, HSP104, YRO2, and SSE2, and more
    • Chemostat cultures allow the growth rate to be controlled. There is the dilution rate D. And a steady state can be reached when volume is held constant.
  • Materials and Methods
    • Strain and growth conditions
      • The strain used, CEN.PK113-7D, was phototrophic and haploid
      • Cultures were grown in a synthetic medium limited by carbon or nitrogen, with a constant dilution rate and stirrer speed.
    • Analytical methods
      • Culture supernatants were collected. Glucose concentrations were analyzed by liquid chromatography. Residual ammonium concentrations were found using cuvette tests and culture dry weights were determined.
      • Trehalose and glycogen measurements were taken.
    • Microarray analysis
      • Cells were taken from the chemostats and probed and hybridized to create microarrays. There were three independent cultures for each growth condition.
      • Microsoft Excel was used for paired analysis. The significance level used for transcript levels was a fold difference of 2 and a false discovery rate of 1%.
      • Venn diagrams and heat map visualizations were made
      • Table 1 shows the characteristics of Saccharomyces cerevisiae grown in ammonium and glucose limited anaerobic chemostats.
  • Results
    • Overview
      • 494 genes yielded significantly different transcription
      • There were 1065 genes that were temperature responsive
      • Only 235 genes were consistent on whether they were up or down regulated. The functions and promoters of these genes were then looked at.
      • Figure 1 is a venn diagram detailing the significant temperature responses of gene transcription.
      • Figure 2 is a heat map to show ratios of the 1065 genes that responded to temperature.
    • Low-temperature chemostat cultivation results in altered uptakes kinetics for limiting nutrient and enhanced catabolite repression
      • In chemostat cultures at 12C the residuals of the growth limiting nutrients glucose and ammonium were higher.
      • Genes involved in growth limiting nutrient intake showed different transcription. HXT3, HXT2, and HXT4 are hexose transporter genes and they showed increases at 12C.
      • Three genes for ammonia permeases MEP1, MEP2, and MEP3 showed reduced differential transcription
      • The increased residuals lead to high catabolite repression. This is seen in the negative regulation of genes for glucose catabolite repression and nitrogen catabolite repression genes.
    • Acclimation to Nonfreezing low temperature does require a high storage carbohydrate content
      • Transcription was induced for genes in the metabolism of stored carbohydrates. But at the steady state 12C chemostat conditions the transcription of these genes was not affected. Some even showed lower transcript levels like TPS1, TPS2, TSL1, GSY1, GSY2, GAC1, and more.
      • Biosynthesis genes for glycogen and trehalose can be induced by stress such as cold shock. This explains why this didn’t show up in steady state conditions.
      • Table 2 shows the protein and carbohydrate storage contents of Saccharomyces cerevisiae
      • Table 3 shows regulation of cis and promoter elements for genes.
    • Up-regulation of the translation machinery at low temperature
      • Some gene involved in making ribosomes had higher transcription at 12C
      • This increase in genes for making proteins didn’t show up in the biomass composition for glucose limited conditions but it did for ammonium limited. The increase in proteins may be because enzymes kinetics are slower at lower temperatures.
      • Figure 3 shows differential expressed of genes in cultures adapted to low temperature.
    • Transcriptional responses to low temperature: adaptation versus acclimation
      • This data was compared to other data sets. There was a group of 259 genes that responded to the temperature change in each compared study. Ninety one were all up regulated and 48 were down regulated in each study.
      • Only 11 genes show consistent regulation between cold shock and steady states and these were YEL073C, YNL024C, YLR225C, PIR3, SFK1, and YPC1.
      • Some lipid metabolism genes were consistently up regulated and some transporter genes were consistently down regulated.
    • Environmental stress response
      • There is a general environmental response in the regulation of these genes. The results showed it as an adaptation response to sudden temperature change.
      • Figure 4 compares the 259 genes that were common among experiments.
      • Figure 5 compares genes that were up or down regulated during acclimation in this study.
      • Figure 6 shows genes that were up or down regulated
  • Discussion
    • Combining experiments can show context independent responses.
    • THe experiment emphasizes the important of distinguishing between adaptation and acclimation.



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