James C. Clements: Week 9

Vocabulary

Terms with (Schade) written after them were inferred from reading the paper "Cold Adaption in Budding Yeast" by Schade et. all 2004.

1. 5'-untranslated region (in regards to secondary mRNA structures): Sequences on the 5' end of mRNA but not translated into protein. It extends from the transcription start site to just before the ATG translation initiation codon. 5' UTR may contain sequences that regulate translation efficiency or mRNA stability. [1]
2. Shine-Dalgarno sequence: ribosomal binding site in mRNA, generally located 8 basepairs upstream of the start codon AUG. [2]
3. NSR1 – encodes nucleolin-like protein involved in pre-rRNA processing and ribosome biogenesis (Schade)
4. TIP1, TIR1,TIR2 (homologue proteins) encode serine and alanin-rich cell wall proteins. May be involved in maintaining cell wall integrity during stress (Schade)
5. nucleolin: A nucleolar protein that functions as a shuttle protein between the nucleus and the cytoplasm and is also found on the cell surface. [3]
6. OLE1 fatty acid desaturase – aids membrane fluidity common among many organisms (both prokaryotes and eukaryotes) (Schade)
7. delta msn2 delta msn4 yeast cells : yeast cells which are lacking in in msn2 and msn4 transcription factors. These transcription factors regulate many stress genes. (Schade)
8. OD600: optical density of a sample measured at a wavelength of 600 nm. [4]
9. hot-phenol method: method of RNA extraction. Should get 300mg of RNA from 2e+8 cells using linked protocol. [5]
10. diauxic: having 2 growth phases [6]
11. open reading frame (ORF): A sequence of DNA triplets, between the initiator and terminator codons, that can be translated into mRNA [7]
12. RNA helicase: Helicases are a class of enzymes vital to all living organisms. They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands (i.e. DNA, RNA, or RNA-DNA hybrid) using energy derived from ATP hydrolysis. [8]
13. oxidative stress: Cell damage caused by free radicals, highly reactive chemicals that contain oxygen and are often produced through normal chemical reactions in the body) [9]
14. RNA polymerase An enzyme that transcribes a DNA sequence, creating mRNA. [10]
15. RNA processing A general term for the maturation of a precursor RNA; includes the processes of RNA splicing, RNA modification, RNA editing and RNA cleavage. [11]
16. ubiquitin-dependent protein degradation: protein degradation dependent on the regulatory polypeptide protein Ubiquitin. [12]
17. basal level: level at point of origin [13]
18. catabolism: breakdown in living organisms of more complex substances into simpler ones together with release of energy [14]

Outline

• What is the main result presented in this paper?
  • The transcriptional responses of budding yest S. cerevisiae to early cold response (ECR) and late cold response (LCR) were determined. The transcriptional response to LCR was similar to that of environmental stress response (ESR) whereas the ECR was seemingly unique.
• What is the importance or significance of this work?
  • This work sheds light on the response of eukaryotic cells to cold environments
• How did they treat the cells (what experiment were they doing?)
  • Initially grown at 30 deg C and then cells kept in early log phase at 10 degrees C for different time intervals.
  • Perform microarray testing on yeast RNA.
• What strain(s) of yeast did they use? Was the strain haploid or diploid?
  • BY4743 (homozygous diploid)
  • BSY25 (homozygous diploid mutant missing MSN2 and MSN4)
• What media did they grow them in? What temperature? What type of incubator? For how long?
  • Yeast Extract Peptone Dextrose medium (YPD): 2% glucose, 2% bactopeptone, 1% yeast extract
  • Grown at 30 degrees dropped to 10 degrees at 4 deg C per minute
  • Grown in 50 ml of medium in 250 ml Erlenmeyer flask shaken at 170 RPM

held at 10 deg C for different time intervals: 10, 30, 130 min, 12 hours, and 60 hours (except for control which was held at 30 deg C for those time intervals).

• What controls did they use?
  • Cy dyes were swapped for reference and experimental samples for different experimental runs
  • Runs were performed in which yeast was kept at 30 deg C and not dropped to 10 deg C
  • Compared results to other similar studies
• How many replicates did they perform per timepoint?
  • 2 replicates for 0, 2, and 12 h. 3 replicates for 10 min, 30 min, and 60 hour. 2 for each with mutant except 3 for 12h
• What mathematical/statistical method did they use to analyze the data?
  • Hierarchical clustering by GeneSpring software based on the matrix of standard correlation from the microarray data.
• What transcription factors did they talk about?
  • Msn2p, Msn4p; these bind to stress response elements
  • Spt23p, Mga2p: ER membrane bound transcription factors that regulate OLE1 (OLE1 is a gene involved in lipid metabolism and is responsible for membrane fluidity.
  • HAP5 and TYE7: carbohydrate metabolism transcription factors induced in LCR
• Briefly state the result shown in each of the figures and tables.
  • Figure 1: Overall look at the transcriptional response to cold for wild-type experiment. A: cluster analysis of microarray data for 634 genes that were determined to be significant. Green: down-regulated, red: induced. Early Cold Response genes shown in sections labeled D and E. A, B, and C show Late Cold Response genes. B and C classify the classification and amount of such classified genes for ECR in B and LCR in C.
  • Figure 2: Comparison of data with that collected by Gasch et. al (2000). A correlation between transcriptional response of ECR from 30 to 10 degrees is found in the shift from 37 deg to 25 deg C as in the Gasch experiment. Not all genes were analogous however, only the ones labeled a and b on the diagram were found to be in correlation.
  • Figure 3: Early Cold Response compared with Late Cold Response, and the genes used in both are compared in Venn Diagrams with the Environmental Stress Response. It is found that the ECR only shares 2 induced genes and 2 repressed genes with the ESR whereas the LCR and the ESR share 87 induced genes and 111 repressed genes. Responses to various drugs are also investigated.
  • Figure 4: Comparison of wild type gene expression for 2 hour and 12 hour time points vs the delta msn2 delta msn4 strains. This data suggests that the ECR expression profile is cold-specific whereas the LCR genes are similar to that of ESR.
  • Figure 5: Investigation of amounts of glycogen (top) and trehalose (bottom) in regards to the wild type and the mutant strain. The mutant strain did not produce a significant amount of trehalose at any time point and produced less glycogen than the wild type at all time points. The mutant strain was less able to accumulate reserve carbohydrates during cold treatment.
  • Figure 6: Comparison of transcriptional response in wild type yeast with the Sahara (2002) study. A common cluster of genes during the LCR was observed, this list includes several general stress-response genes. The data for the ECR varied drastically, however between the two data sets. The 2002 Sahara paper indicated that the ribosomal genes would be induced whereas the Schade paper noticed a decrease in transcript abundance. This could be explained by difference in yest strain or in the phase that the yeast was grown in.
• Closing Remarks
  • Cold response happens in 2 phases: early and late cold response. In this experiment, the late cold response began sometime after 2 hours.
  • Early cold response is fairly unique to cold
  • Late cold response is similar to environmental stress response
  • More data must be collected in order to fully understand this phenomenon. Part of the response seems to also depend on what growth phase the yeast is in.