Elizabeth Polidan Week11

Elizabeth Polidan

BIOL 398.03 / MATH 388

• Loyola Marymount University

• Los Angeles, CA, USA

• Email me through OpenWetWare

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Vocabulary

1. trehalose
   • A nonreducing disaccharide that acts as a storage carbohydrate and protects cells against various environmental stress conditions.
   • Charlemagne-Gilles, H., et al., Role of trehalose in survival of Saccharomyces cerevisiae under osmotic stress, Microbiology (1998), 144, pp. 671-680. (http://mic.sgmjournals.org/content/144/3/671.full.pdf)
2. mannoproteins
   • Yeast cell wall components composed of proteins with mannose groups attached.
   • http://www.biology-online.org/dictionary/Mannoproteins
3. prototrophic
   • A strain that has the same nutritional requirements as the wild strain from which it is derived.
   • http://www.biology-online.org/dictionary/Prototrophic_strains
4. fatty-acid desaturase
   • Enzymes that put double bonds into the hydrocarbon areas of fatty acids.
   • http://www.biology-online.org/dictionary/Desaturase
5. cis-regulatory motifs
   • A region of DNA or RNA that regulates the expression of genes also located on that same strand of DNA or RNA.
   • http://en.wikipedia.org/wiki/Cis-regulatory_element
6. HXT5
   • Hexose transporter with moderate affinity for glucose.
   • http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=HXT5
7. HXT6
   • High-affinity glucose transporter
   • http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=hxt6
8. Carbon recovery
   • Ratio of amount of recovered carbon to the amount available at the onset of fermentation.  It is a measure of efficiency.  Usually percentage reflecting # moles produced per 100 moles of substrate used. 
   • El-Mansi, E.M.T, Bryce, C.F.A., Demain, A.L., and Allman, A.R. editors (2006) Fermentation Microbiology and Biothechnology, 2nd edition, Boca Raton, FL: CRC Press, p. 31.
9. rRNA
   • Ribosomal RNA
   • http://www.biology-online.org/dictionary/Rrna

Outline of Tai et al. (2007)

1. Introduction
   • A study of differential transcriptional regulation at low temperature was performed using chemostat cultivation rather than batch cultures in order to eliminate any effects associated with differential specific growth rates.
   • This contrasts the role of genes in adaptation to rapid transition to cold temperature (cold shock) with their role in longer term acclimation to a low temperature environment.
   • Contextual dependency of response to lower temperatures
     • Responses may be due to specific growth rates rather than temperature
     • Responses may be due to specific place in a hierarchical regulation scheme
2. Methods
   • Experimental setup
     • Used CEN.PK113-7D (MATa), a strain of S. cerevisiae that has the same nutrient requirements as the wild strain.
     • Cultures were grown in 2 liter chemostats at 12oC and 30oC.
     • The dilution rate was 0.03 per hour for both temperatures.
     • The media was a synthetic media with all required nutrients (save glucose and ammonium) in excess. At each temperature there was a nitrogen limited (glucose in excess) culture and a glucose limited (nitrogen in excess) culture.
     • To avoid issues of temperature-based oxygen solubility differences the system was maintained anaerobically.
     • The pH was maintained at 5.0.
   • Measurements
     • Culture dry weights and whole cell proteins were measured after steady state was reached.
     • Concentrations of glucose and metabolites were measured.
     • Trehalose was measured using three measurements for each chemostat.
     • Glycogen was measured using two measurements for each chemostat.
     • Microarrays were created for each temperature and nutrient combination.
3. Analysis
   • Analysis of experimental data
     • The MS Excel add-in SAM (significance analysis of microarrays) was used for pair-wise comparisons.
     • Visualizations (Venn diagrams and heat map) were created using Expressionist Analyst.
     • Web-based tools were used for analysis of overrepresentation and sequence analysis.
   • Comparison with batch culture data
     • Batch culture data from three other experiments (from three different teams) was used to compare with the chemostat culture results.
4. Discussion
   • A comparison of the gene expression 12oC vs 30oC (figure 1)
     • In the nitrogen limited system 571 genes showed significant up or down regulation.
     • In the carbon limited system 259 genes showed significant up or down regulation.
     • There were 235 genes that showed significant differences in gene expression in both the nitrogen and carbon limited systems.
   • Figure 2 shows heat map representing the transcription level ratio of the genes that were significantly up or down regulated. The genes are grouped by nutrient limitation and by up vs down.
   • The specific growth rate of 0.03 / h is ~75% of mumax at 12C, as opposed to 10% of mumax at 30C. This results in an increased concentration of the residual nutrients at the lower temperature. An increased concentration of the residual nutrients results in catabolite represession.