Alison S King Week 3

Purpose

The purpose of this assignment is to get comfortable reading and understanding primary research articles as well as presenting them in a Journal Club. This assignment also teaches us important information about transcriptional responses to cold shock.

Biological Terms and Definitions

phosphorylation: A process in which a phosphate group is added to a molecule, such as a sugar or a protein. (National Cancer Institute, 2019)
diauxic shift: The switch from rapid fermentative growth in the presence of a rich carbon source to slower exponential growth by aerobic respiration using ethanol once the preferred carbon source has been exhausted. (Stanford University, 2019)
trehalose: A disaccharide formed by a (1<->1)-glycosidic bond between two units of D-glucose. (Stanford University, 2019)
prehybridization: A procedure used for the treatment of nitrocellulose or nylon membranes following Northern or Southern transfer and before the use of labelled nucleic acid probes to detect specific sequences on the blot. The intention is to block the surface of the membrane to decrease non‐specific binding of the probe. A variety of blocking agents can be used. (Cammack R. et al., 2008)
centrifugation: An apparatus in which fluids may be rotated rapidly so that substances (solutes or dispersed particles) of different densities may be separated by centrifugal force. (Cammack R. et al., 2008)
desaturase: General name for any enzyme catalysing a desaturation reaction. (Cammack R. et al., 2008)
vesicle: A closed structure, found only in eukaryotic cells, that is completely surrounded by a unit membrane but, unlike a vacuole, contains material that is not (or is not known to be) in the liquid state. (Cammack R. et al., 2008)
helicase activity: Catalysis of the reaction: NTP + H2O = NDP + phosphate, to drive the unwinding of a DNA or RNA helix. (Stanford University, 2019)
glycogen: A polydisperse, highly branched glucan or polyglucose, composed of chains of d‐glucose residues in α(1→4) glycosidic linkage, joined together by α(1→6) glycosidic linkages; a small number of α(1→3) glycosidic linkages and some cumulative α(1→6) links also may occur. Its structure is similar to that of amylopectin but with more, though rather shorter, branches; those in glycogen variously contain 8 to 12 glucose residues. The size of the molecule varies greatly according to source and method of measurement, with average Mr values of 2.5 × 105 to upwards of 1.5 × 107 reported. Glycogen serves as a cellular store of glucose; it occurs, frequently as granules, in all animal tissues, especially liver and skeletal muscle, and also in some bacteria and yeasts. The glycogen molecule is linked to the protein glycogenin which is required for the ab initio synthesis of glycogen by glycogen synthase. (Cammack R. et al., 2008)
diploid: Describing a cell or nucleus having two sets of homologous chromosomes; i.e. containing twice the haploid number. (Cammack R. et al., 2008)

Outline of Article

Main Result

The growth rate of S. cerevisiae cells decreased significantly when they were subjected to cold shock, whereas the growth curve remained normal.
This ability to adapt to cold temperatures could be attributed to changes in gene expression.
- Transcriptional cold response made of two distinct expression patterns (early and late phases)
  - Early: changes in membrane fluidity, destabilization of RNA secondary structures for more efficient protein translation
  - Late: environmental stress response, result of misoflded proteins, reduced enzyme activities, decreased transport caused by cold

Significance of this work

It is important for organisms to be able to adapt to the environment and deal with changing conditions (i.e. cold shock)
Study shows how yeast can adapt through changes in gene expression
- Possible to apply to other more complex organisms in the future with further research

Limitations of previous studies

Heat shock study: yeast cells induce heat shock proteins that allow them to fix damaged proteins
Studies on other stresses: transcription changes in a common set of genes, called the general stress response
Not much was previously studied about the response to cold temperatures
- Cold causes changes in physical and biochemical properties
- Wanted instead to look at transcriptional response to cold and compare to responses to other stressors

The Experiment

Treatments

10 degrees Celsius (experimental) or 30 degrees Celsius (control)

Strains of yeast

BY4743 (MATa/α, wild-type)
BSY25 (BY4743, except homozygous Δmsn2::kanMXΔmsn4::kanMX met15)
For growth curve experiments: W303 (MATa/α, wild-type)
Diploid strains

Culture conditions

Grown in YPD medium
- Inoculated and grown overnight at 30 degrees Celsius
- Then, diluted, grown at 30 degrees C, shaken, and transferred to 10 degree C water bath shaker.
- Then, incubated for 10, 30, 120 min, temperature decreasing 4 deg C per min
- Cultures reached 10 or 30 (control) deg C and then frozen/stored at -80 deg C

Controls

Cultures ensured to be in same physiological state prior to experiment
Consistent time points (0, 2, 12 hours; 10 min, 30 min, 60 hours)
- Performed two replicates of 0, 2, 12 hours (three for 12 h)
- Performed three replicates of 10 min, 30 min, 60 hours
The control groups were kept in 30 degrees C

Method of preparing RNA

Isolated RNA with hot-phenol method
- Cells processed by extracting with phenol twice (10 min each), added glass beads for the 60 hour
Labeled mRNA with Cy3 and Cy5-dCTP by reverse transcription
- This process gave them cDNA which was then hybridized onto DNA microarrays
  - 20:1:1 DigEasyHyb solution, yeast tRNA, sonicated salmon sperm DNA, for 2 hours at 42 deg C
  - Microarrays washed twice for 2min and dried and then hybridized

Data Analysis

Statistical Methods

Hierarchical clustering of genes
Used statistical analysis and visualization to interpret the raw data
- GeneSpring software
Data available from [:http://cbr-rbc.nrc-cnrc.gc.ca/genetics/cold/]

Figures

Figure 1: Transcriptional response to cold.
- (A) Hierarchical cluster of wild-type incubated at 10 deg C (634 genes that showed significant difference)
  - Color determined by ratio of transcription (experimental over reference)
  - Horizontal axis is genes (similarities shown by dendrogram)
  - Vertical axis is time exposure to cold
  - A, B, C = late cold response
  - D, E = early cold response
- (B, C) Classification of ECR and LCR genes
  - Distributions of functional categories of genes (horizontal)
  - Vertical axis is number of genes in each category
- This figure showed that genes did respond to the cold and induce transcription.
Figure 2: Transcriptional profiles of early cold response during temperature downshifts.
- x-axis = time points when temperature was brought from 37 to 25 deg C
- y-axis = genes, a and b correspond to genes with correlating responses
- Comparing their published data (30 to 10 deg C) with unpublished (37 to 25 deg C)
Figure 3: Comparison of the transcriptional responses to cold and other environmental stresses.
- Side-by-side comparison of genes' transcription in cold shock versus other stresses (i.e. heat shock)
  - (A) ECR, (B) LCR
- (C) Showing overlap between LCR and ESR, and ECR and ESR
  - Significant for LCR, not for ECR
Figure 4: Regulation of gene expression during cold treatment.
- 634 cold-responsive genes clustered based on expression patterns
  - Expression ratio is average of all experiments
- 78% of LCR genes unaffected without Msn2p/Msn4p, so may be other transcription regulators involved
- No difference between wild-type and those without Msn2p/4p, so ECR genes are regulated by something else
Figure 5: Accumulation of reserve carbohydrates during cold treatment.
- Time vs. amount of glycogen or trehalose
  - Amounts are averages of the three experiments
- No accumulation of glycogen or trehalose in first two hours, but increase in both after 12 hours of cold
  - Agrees with microarray data (genes for carbohydrate metabolism are induced at this time)
  - Even more glycogen and trehalose after 60 hours
Figure 6: Comparison of the transcriptional response to cold observed in this study to that reported by Sahara et al. (2002).
- Side-by-side of the two studies' results
- Genes clustered by transcriptional profile, each line is a gene, color depends on the change in transcription in the cold
- The close-ups show the differences and similarities between the two studies
  - Both showed increased transcription in certain genes in response to cold

Implications of the Study

Compared to other studies, this work focused more in depth on the cold shock response
- Most others looked at other environmental factors
- Some of the others did look at cold shock briefly, and not all data agrees
This study helps us understand the transcriptional response to cold
- Important for survival and growth in cold
- Important for understanding and promoting cellular processes
- Could we extend these results to other things besides yeast?
  - Might be useful for humans or animals
In the future, could look at the specific key regulatory mechanisms that promote survival in cold
- Again, could extend to other organisms?

My Evaluation

The authors supported their claims well with data, figures, and explanations.
- The figures were convincing in showing that specific genes had transcriptional responses to cold shock, etc.

Acknowledgements

Ava Lekander, my partner for this assignment, and I texted a couple of times over the course of the week to ask and answer questions about the article outline. We also met one time outside of class to discuss our Journal Club presentation and go over our assigned figures (4 and 5).

Except for what is noted above, this individual journal entry was completed by me and not copied from another source.

Alison S King (talk) 18:10, 6 February 2019 (PST)

References

Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (2008) Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press.

National Cancer Institute (n.d.) NCI Dictionary of Cancer Terms. Retrieved from https://www.cancer.gov/publications/dictionaries/cancer-terms/ on 5 February 2019.

Stanford University (n.d.) Saccharomyces Genome Database. Retrieved from https://www.yeastgenome.org/ on 5 February 2019.

Loyola Marymount University (5 February 2019) BIOL388/S19:Week 3. Retrieved from https://openwetware.org/wiki/BIOL388/S19:Week_1 on 5 February 2019.

Schade, B., Jansen, G., Whiteway, M., Entian, K. D., & Thomas, D. Y. (2004). Cold Adaptation in Budding Yeast. Molecular Biology of the Cell, 15(12), 5492-5502.

Links

Alison S King

Class Page: BIOL388/S19

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