Alyssa N Gomes Week 10 Journal

Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analysis

10 Vocab Words

• Mannoprotein: Yeast cell wall components that are proteins with large numbers of mannose groups (mannose: ) attached. They are antigenic, meaning that they have the ability to combine specifically with the final products of the immune response.
  • http://www.biology-online.org/dictionary/Mannose
• Trehalose: A white, crystalline disaccharide, C12 H22 0 11, occurring in yeast, fungi bacteria, etc.
  • http://dictionary.reference.com/browse/trehalose
• Prototrophic:having the nutritional requirements of the normal or wild type
  • http://www.merriam-webster.com/dictionary/prototrophic
• Cryostat:an apparatus for maintaining a very low temperature.
  • http://dictionary.reference.com/browse/cryostat
• Supernatants:The soluble liquid fraction of a sample after centrifugation or precipitation of insoluble solids.
  • http://www.biology-online.org/dictionary/Supernatant
• Chromatin immunoprecipitation: A procedure used to determine whether a given protein binds to or is localized to a specific DNA sequence in vivo.
  • http://www.bio.brandeis.edu/haberlab/jehsite/chIP.html
• cis-regulatory: A noncoding DNA sequence in or near a gene required for proper spatiotemporal expression of that gene, often containing binding sites for transcription factors
  • http://www.nature.com/scitable/definition/cis-regulatory-element-cis-regulatory-element-75
• Permease: A membrane protein that increases the permeability of the plasma membrane to a particular molecule, by a process not requiring metabolic energy.
  • http://www.biology-online.org/dictionary/Permease
• Homeoviscous:This compositional adaptation of membrane lipids, called homeoviscous adaptation, serves to maintain the correct membrane fluidity at the new conditions.
  • http://www.encyclo.co.uk/meaning-of-Homeoviscous%20adaptation
• First-Order Kinetics:An order of chemical reaction in which the rate of the reaction depends on the concentration of only one reactant, and is proportional to the amount of the reactant.
  • http://www.biology-online.org/dictionary/First-order_kinetics

Outline of "Acclimation of Saccharomyces cerevisiae to Low Temperature: A Chemostat-based Transcriptome Analysis"

*Introduction

• • Saccharomyces cerevisiae is a yeast that is exposed to many external and environmental changes that affect processes and chemical structures.
  • This paper will focus on the effect of cold temperatures on the cultures
    • There is a difference in sudden exposure vs gradual exposure because sudden exposure will respond with shock and stress-related responses, whereas gradual exposure will lead to acclimation
  • Previous studies have shown a difference in the early cold response (<1 hour) and late cold response (>12hours)
  • Although this has priorly been studied, some questions remain unanswered:
    • Why did prior studies done by Sahara, Homma , Schade, Murata differentiate in their answers about the growth of expression ribosomal protein genes?
    • Why did cold shock bring out reserve carbohydrate while trehalose only arose in desperate freezing conditions?
    • Is there a way to bring out the Msn2p/Msn4p complex that has been previously suggested to be a transcriptional factor in cold weather?
    • How can we study and describe the difference between acclimation and shock responses in S cerevisiae?
  • Chemostat cultures will allow many factors to remain stable in acclimatized environments
  • Goal: study the steady-state acclimatized growth of this yeast culture and its transcription, under temperatures of 12-30 degrees celsius and at a growth rate of 0.03 h^-1

*Materials and Methods

• • Strain and Growth Conditions
    • Strain: Saccharomyces cerevisiae
    • Growth rate: 0.03 h^-1
    • Volume: 1.0 l
    • Temperature probe set to 12 degrees Celsius initially
    • Anaerobic growth, biomass dry weight, metabolites, dissolved oxygen and steady-state set stable
  • Analytical Methods:
    • Supernatants were collected with rapid sampling
    • Liquid chromotography was used to analyze concentrations of glucose and metabolites on an AMINEX HPX-87H ion exchange column using 5 mM H2SO4
    • Cuvette tests from DRLANGE were used to examine the residual ammonium concentrations
    • Triplicate measurements in the chemostate measured trehalose
    • Roche kit no. 0716251 examined the amount of glucose released from glycogen and trehalose in breakdown methods
  • Microarray Analysis:
    • Results for the listed growth conditions came from three separately cultured replicates.
    • The coefficients of variation for the four growth conditions was 0.20
    • The ACT1 transcript varied by less than 12% for the four growth conditions
    • Microsoft Excel was used in order to run significance analyses on microarray add-ins
    • The analyses used a threshold fold difference of 2 and median false rate discovery of 1%
    • Venn diagrams and heat maps were used with Expressionist Analyst version 3.2
    • Regulatory Sequence Analysis (RSA) Tools were used to examine the promoter values
    • the Database for Annotation, Visualization and Integrated Discovery (DAVID) was used in order to assess overrepresentation
    • Overrepresentation of transcription-factor binding sites as defined by chromatin immunoprecipitation was calculated with Fisher's exact test, a Bonferroni correction, and a p-stat
    • The data has been submitted to the Genome Expression Omnibus database under the number GSE6190
  • Comparison with Other S. cerevisiae Low-Temperature Transcriptome Datasets
    • Batch transcriptome genomes used in this for comparison studies are: Beltran et al., Murata et al., Sahara et al., Schade et al., and Gasch et al.
    • The collection of stress-response genes were found on: http://genome-www.stanford.edu/yeast_stress/

*Results

• • Overview
    • The culture, grown at 12 and 30 degrees celsius was under 0.03 h^-1 growth for its culture, allowing for steady-state growth
    • Cultures were grown anaerobically, in order to decrease external factors
    • In both 12 degree and 30 degree cultures, both carbon and nitrogen limiting cultures had the same biomass, indicating no effect on growth efficiency
    • In glucose limiting cultures, 494 genes showed significantly different transcription levels
    • In nitrogen limiting cultures, 806 genes showed significantly different transcription levels
    • The total number of temperature responsive genes was 1056, 16% of the total genes
    • 235 of the genes showed a consistent increase/decrease in transcript levels, no matter the limiting factor
    • In determining the responses for regulatory networks to temperature acclimation, temperature-responsive genes were evaluated under the screenings in order to seek changes in enrichment of nutrients and promoter regions
  • Low-Temperature Chemostat Cultivation Results in Altered Uptake Kinetics for the Limiting Nutrient and Enhanced Catabolite Repression