William A. C. Gendron Week 10

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

1. Definitions
   • Trehalose(also called mycose): This is "a non reducing disaccharide" which in this context it is being used as a cryoprotectant as it is brought to near freezing conditions. It can also act as a preservative and an anti-oxidant. TPS1 and TPS2 are responsible for its biosynthesis in yeast. Source: http://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-19924?rskey=ZqLfPq&result=1
   • Mannoproteins: These are proteins "that contain 15 to 90% mannose by weight" Sources: http://link.springer.com/chapter/10.1007%2F978-3-642-68234-6_18
   • Desaturase: An enzyme that catalyzes desaturation. Desaturation is when "two hydrogen atoms or a hydrogen and a hydroxyl group are removed from adjacent atoms." Source: http://www.oxfordreference.com/view/10.1093/oi/authority.20110803095712459 http://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-4993?rskey=k9Vxrx&result=1
   • Permease: A membrane transport protein in or out of the cell. There really should not be two names for this. Source: http://www.oxfordreference.com/view/10.1093/oi/authority.20110803100318301
   • Zero trans-influx transport assay: I could not find an exact definition to how they do this but it measures the rates of glucose uptake in this context. None of the sources I found really defined how it was done even when I did a wider google search. Source: http://link.springer.com/article/10.1007/s00253-002-1085-6
   • Homeoviscous: A cell will alter the number of long or saturated fatty acids in its cell membrane to adjust for the temperature. Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC388039/
   • DEAD Box Family: This refers to 4 amino acids that form in this order: -D-E-A-D- (Asp-Glu-Ala-Asp) and this acts as an ATP binding site. Source: http://www.oxfordreference.com/view/10.1093/oi/authority.20110803095704770?rskey=nZKYQA&result=2
   • First-Order Kinetics: This is a chemical reaction that is only dependent on one of the reactants. Source: http://www.biology-online.org/dictionary/First-order_kinetics
   • Cis-regulatory: This is a sequence near the gene being referenced, which acts as a binding site for transcription factors. It may be in or near the gene. Source:http://www.bio.brandeis.edu/haberlab/jehsite/chIP.html
   • Chromatin immunoprecipitation: Chromatin immunoprecipitation, or ChIP, refers to a procedure used to determine whether a given protein binds to or is localized to a specific DNA sequence in vivo. The diagram below illustrates the basic steps of this procedure. Source: http://www.nature.com/scitable/definition/cis-regulatory-element-cis-regulatory-element-75

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

• Introduction
  • This research used Saccharomyces cerevisiae as the model organism.
  • Focused on discovering the true genes associated with cold shock reaction.
  • Batch based experiments create a unique situation that is not just cold shock stress.
  • Prior studies show discrepancies in ribosomal protein expression. Why is that?
  • Discrepancies between carbohydrates and trehalose usage.
  • Further explore the Msn2p/Msn4p complex.
  • Evaluate adaptation and acclimation studies.
  • A chemostat is a unique way to study yeast: it supplies a stable environment that can be constantly growing. Changes growth rate and other features.

• Materials and Methods
  • Saccharomyces cerevisiae was was grown at 12 and 30 degrees Celsius. Growth rate for the 12 degrees was .03h^-1 with a volume of 1.01 liters. Set ups of nitrogen and carbon limited chemostats.
  • Basic Analysis:
    • Supernatants were collected to do liquid chromatography and other tests. This evaluated the level of glucose and metabolites.
    • Ammonium concentration was evaluated with a similar chromatography method.
    • Glucose creation from glycogen and trehalose was evaluated by a Roche kit.
  • Microarray analysis:
    • Measured three times and used Microsoft Excel to run the statistics.
    • Used two-fold difference as a threshold and calculated a median false rate at 1%.
    • Evaluated overrepresentation with DAVID(Database for Annotation, Visualization and Integrated Discovery) along with a Fisher's exact test, a Bonferroni correction and a p-test.
    • Compared data with data from Beltran et al., Murata et al., Sahara et al., Schade et al., Gasch et al. and the Stanford Yeast Stress Database.

• Results
  • Low-Temperature Chemostat Cultivation Results in Altered Uptake Kinetics for the Limiting Nutrient and Enhanced Catabolite Repression
    • More residual nutrients in 12 degree than 30. Reduced transport and metabolism is the root of this.
    • Hexose transport genes increased transcription, in theory to account for the decreased rate of transport in the 12 degree.
    • HXT5 and 16 increased in 30 degree.
    • In nitrogen limited cultures, permeases that targeted ammonia changed. MEP3 increased for 12 degree while high affinity MEP1 and 2 were reduced.
  • Acclimation to Nonfreezing Low Temperature Does Not Require a High Storage Carbohydrate Content
    • Transcription was not changed in genes for trehalose and glycogen metabolism.
    • Carbohydrate synthesis decreased at lower temperatures.
  • Up-Regulation of the Translation Machinery at Low Temperature
    • Higher amounts of transcription seen in 16 of the ribosome biogenesis genes at 12 degrees(both nitrogen and carbon limited).
    • 12 degrees nitrogen and carbon limited showed evidence of much higher rRNA and protein than 30.
  • Transcriptional Responses to Low Temperature: Adaptation versus Acclimation
    • Compared to previous studies
    • Found that our of 259 genes, 139 matched up or down regulation across cold shock genes.
    • Specific genes were identified to be highly consistent.
  • Context Dependency of Temperature Response
    • They describe why this has better controls than other experiments.
    • Better O2 control than flask batches
    • Found genes that did not decrease with temperature but use to be linked to other experiments that suggested that they were.
  • Environmental Stress Response, a Low-Temperature Adaptation-specific Response
    • Compared to database of genes that react in environmentally stressful situations
    • About 1/3 of the genes from this study were found to be linked to ESR.

• Discussion
  • Chemostat-based Low-Temperature Transcriptomics: Experimental Design
    • Explains that there are too many variables to control for and multiple experiments are required to test the limits.
    • Requires multiple tests to get a full picture of the genes.
  • Specific Growth Rate and ESR
    • Growth rates between temperature groups were quite different.
    • ESR genes were found to make up a portion of the genes activated.
  • Transcriptional Responses to Low Temperature: Acclimation versus Adaptation
    • Confusing terms but it focused on the timing of different genes. I would have preferred another word instead of adaptation, because I think of it as an evolutionary term.
    • It wrapped up saying that that this alternative system can be used to parse out genes that are caused by the batch system.

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